Biology of Blood and Marrow Transplantation
Volume 13, Issue 7 , Pages 853-862, July 2007

Cyclophosphamide following Targeted Oral Busulfan as Conditioning for Hematopoietic Cell Transplantation: Pharmacokinetics, Liver Toxicity, and Mortality

  • Jeannine S. McCune

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington School of Pharmacy, Seattle, Washington
    • Corresponding Author InformationCorrespondence and reprint requests: Jeannine S. McCune, PharmD, Department of Pharmacy, Box 357630, University of Washington, Seattle, WA 98195.
    • ,
  • Ami Batchelder

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
    • ,
  • H. Joachim Deeg

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington School of Medicine, Seattle, Washington
    • ,
  • Ted Gooley

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
    • ,
  • Scott Cole

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
    • ,
  • Brian Phillips

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
    • ,
  • H. Gary Schoch

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
    • ,
  • George B. McDonald

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
    • University of Washington School of Medicine, Seattle, Washington

Received 25 January 2007; accepted 29 March 2007.

Article Outline

Abstract 

The pharmacokinetics of cyclophosphamide (CY) and its metabolites hydroxycyclophosphamide and carboxyethylphosphoramide mustard were determined in 75 patients receiving targeted oral busulfan followed by i.v. CY (TBU/CY) and in 147 patients receiving i.v. CY followed by total body irradiation (CY/TBI) in preparation for hematopoietic cell transplantation (HCT). In the TBU/CY patients only, the association of the pharmacokinetic data with liver toxicity, relapse, and survival was evaluated. CY was infused at 60 mg/kg/day over 1 or 2 hours on 2 consecutive days; the majority of patients had BU levels targeted to a steady state plasma concentration (Css) of 800-900 ng/mL. Systemic exposure (i.e., area under the concentration-time curve [AUC]) of CY, hydroxycyclophosphamide, and carboxyethylphosphoramide mustard was measured. Liver toxicity was assessed as the development of hepatic sinusoidal obstruction syndrome (SOS). CY metabolism was highly variable and age dependent. TBU/CY-treated patients had lower AUCCY (P < .0001), higher AUCHCY (P < .0001), and higher AUCCEPM (P = .15) than CY/TBI-conditioned patients. Among patients receiving TBU/CY, 17 (23%) developed SOS, and there were no statistically significant associations between the AUC of CY or its metabolites and SOS, nonrelapse mortality, relapse, or survival (all P >.15). In conclusion, CY exhibits conditioning-regimen dependent pharmacokinetics and pharmacodynamics, suggesting that lowering CY doses is unlikely to improve outcomes to TBU/CY. Alternative strategies, such as administering i.v. busulfan or CY before BU, should be explored.

Key Words: Hematopoietic cell transplant, Myeloablative regimen, Busulfan, Cyclophosphamide, Diphenylhydantoin, Pharmacokinetics, Sinusoidal obstruction syndrome, Mortality, Survival

 

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Introduction 

A myeloablative conditioning regimen combining busulfan and cyclophosphamide (BU/CY) is frequently used to prepare patients for hematopoietic cell transplantation (HCT) [1]. Several reports suggested that sinusoidal liver toxicity was associated with elevated BU exposure in patients receiving high dose BU (1 mg/kg/dose orally every 6 hours for 16 doses) followed by CY [2, 3, 4]. Specifically, liver toxicity was observed with a BU area under the plasma-concentration time curve (AUC) >1250 [2] or 1500 μM·h [3, 4]. These correspond to a BU steady-state concentration (Css) of 925 or 1025 ng/mL, respectively, when BU is dosed every 6 hours for 16 doses. This association was observed predominantly in adult populations receiving HCT for various diseases. On the other hand, studies in patients with chronic myelogenous leukemia (CML) in the chronic phase who received BU/CY showed that BU Css >900 ng/mL did not correlate with liver toxicity, but was inversely related to the risk of leukemic relapse [5, 6]. In vitro studies with murine hepatocytes revealed that BU depletes hepatic glutathione and at high concentrations induces oxidative stress [7]. Many HCT teams adjust BU doses to a target Css to minimize the risks of liver toxicity, rejection, and relapse [8]. In combination with CY, BU administered intravenously (i.v.) is associated with lower rates of liver toxicity than when given orally when the drug is dosed by body weight (i.e., not targeted) [9, 10]. Liver toxicity occurs in 5% [9] to 18% [10] of patients receiving BU/CY with i.v. BU dosed by body weight (0.8 mg/kg i.v. every 6 hours for 16 doses). Thus, regimen-related toxicity has remained a problem in patients conditioned with targeted oral BU/CY (TBU/CY) [6, 11, 12] and with weight-based dosing of i.v. BU [9, 10, 13].

As targeting BU doses minimized interpatient variability in exposure, we hypothesized that regimen-related toxicity in TBU/CY conditioned patients was related to variability in CY metabolism. In vitro studies of isolated hepatocytes and sinusoidal endothelial cells incubated with CY showed that sinusoidal endothelial cells were highly sensitive to CY metabolites generated by hepatocytes, consistent with the clinical picture of sinusoidal liver toxicity [14]. The AUC of i.v. CY is more variable than that of oral or i.v. BU [15, 16, 17]. Higher exposure to the CY metabolite carboxyethylphosphoramide mustard (CEPM) was strongly correlated with liver toxicity and mortality in 147 patients conditioned with a regimen of CY followed by total body irradiation (TBI), that is, 23 patients (16%) developed moderate or severe sinusoidal obstruction syndrome (SOS) [15]. Exposure to CEPM correlated statistically significantly with the development of SOS, nonrelapse mortality (NRM), and survival, after adjusting for age and irradiation dose. There was no significant correlation with engraftment and tumor relapse. These in vitro and in vivo data suggest that metabolites of CY are toxic to hepatic sinusoids, thereby causing SOS. Accordingly, we evaluated the pharmacodynamic relationship between the exposure to CY and 2 of its metabolites, hydroxycyclophosphamide (HCY) and CEPM, with clinical outcome in patients conditioned with TBU/CY. As CY pharmacokinetics have been found to be dependent upon the type of conditioning regimen [18], we also compared the pharmacokinetics of CY, HCY, and CEPM between patients who received TBU/CY (i.e., CY following BU) and patients (previously reported) who received CY/TBI (i.e., CY first) [15].

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Materials and Methods 

Patient Selection 

Patients 18 to 65 years of age were eligible for study participation if they had a diagnosis of myelodysplastic syndrome, myelofibrosis, or another hematologic malignancy for which a transplant conditioning regimen of TBU/CY was appropriate. Related patient/donor pairs were HLA identical by high resolution DNA typing. Unrelated donors were selected on the basis of high-resolution HLA typing as described [19, 20]. Written consent was obtained using forms approved by the institutional review board of the Fred Hutchinson Cancer Research Center.

Patient, disease, and transplant characteristics of the cohort receiving TBU/CY conditioning are summarized in Table 1. Seventy-five patients, 20-66 years of age, were conditioned with TBU/CY (49 with myelodysplastic syndrome [MDS], 20 with acute myelogenous leukemia [AML], and 6 with CML). Hematopoietic cell donors were family members for 42 patients and unrelated individuals for 33 patients.

Table 1. Patient, Disease, and Transplant Characteristics of Patients Receiving TBU/CY
Number of patients75
Agea (years)47 ± 11
(20-66)
Men:women41:34
Diagnosisb
Myelodysplastic syndrome
Refractory anemia30(40%)
Refractory anemia with excess blasts10(13%)
Myelofibrosis4(6%)
Chronic myelomonocytic leukemia3(4%)
Refractory anemia with ring sideroblasts2(3%)
Acute myelogenous leukemia20(26%)
Chronic myelogenous leukemia6(8%)
Donorsc
Related
HLA-identical sibling38(51%)
HLA-nonidentical family memberc4(5%)
Unrelated33(44%)
Antifungal drugs administered with cyclophosphamideb
Fluconazole56(75%)
Voriconazole vs. fluconazole12(16%)
Other7(9%)

aMean ± standard deviation (range).

bData are reported as number (percentage) of participants.

cHLA nonidentical sibling in three and parent in one patient.

The patient, disease, and transplant characteristics and the pharmacodynamics of CY and metabolites of the cohort receiving CY/TBI conditioning have been previously published [15].

Conditioning Regimen 

The conditioning regimen of TBU/CY consisted of oral BU, 1 mg/kg administered every 6 hours (total of 16 doses, adjusted for plasma target levels as described below) followed by i.v. CY, 60 mg/kg administered every 24 hours for 2 doses. In a subset of patients (N = 21), thymoglobulin was given, starting on the same day as CY and continued over 3 days for cumulative doses of 4.5 or 6 mg/kg [12]. Donor hematopoietic cells were infused 48 hours after the last dose of CY (day 0).

BU doses were adjusted to a target Css as previously described [2]; the target Css was 800-900 ng/mL for 69 patients, 900-1000 ng/mL for 1 patient (with AML) and >900 ng/mL for 5 patients (all with CML). Blood sampling for BU pharmacokinetics occurred after doses 1, 5, and 9 (at 0, 30, 60, 90, 120, 240, 300, and 360 minutes after dose 1; and at 0, 60, 120, 240, and 360 minutes after doses 5 and 9). Rapid quantitation of BU plasma concentrations was achieved by gas chromatography-mass spectrometry, with a concentration range of 25 to 4500 ng/mL and interday coefficient of variability of <8%. Determination of the Css and clearance of oral BU was calculated by noncompartmental methods using WinNonlin (Pharsight, Mountain View, CA). The estimate of BU clearance (dose/AUC) was used to calculate the dose required to achieve the desired Css (clearance × target Css × time between doses). BU doses were adjusted within 3 hours of the last pharmacokinetic sample (i.e., the Css estimated from dose 1 was utilized for dose 3). Average BU Css was calculated as the mean dose/(mean clearance)(dosing interval). The mean clearance was calculated as the mean of these values measured after doses 1, 5, and 9.

Seizure prophylaxis consisted of phenytoin given as a loading dose of 10-15 mg/kg at least 6 hours before BU administration, followed by maintenance doses of 300 mg orally once daily until 24 hours after the final BU dose.

CY was infused through a central venous access catheter over 1 or 2 hours at doses of 60 mg/kg adjusted ideal body weight per day on 2 consecutive days. The infusion duration followed Fred Hutchinson Cancer Research Center Standard Practice Guidelines. Specifically, total CY doses of <5000 mg were infused over 1 hour, and CY doses equal to or >5000 mg were infused over 2 hours. The first CY dose was administered 12-15 hours after the last oral BU dose. CY doses were fixed; no adjustments were made. During the days of CY infusion, patients received MESNA (2-mercaptoethane sulfonate) prophylaxis for uroepithelial protection at milligram doses equal to those of CY.

The use of all antifungal agents administered concomitantly with the conditioning regimen was recorded because some prophylactic antifungal agents (e.g., fluconazole and itraconazole) inhibit cytochrome P450 (CYP) activity and may influence CY pharmacokinetics [21]. Antifungal prophylaxis given concomitantly with CY administration consisted of fluconazole in 56 patients; 12 patients were enrolled in a blinded, randomized trial comparing fluconazole to voriconazole, 5 received miscellaneous antifungal prophylaxis (2 received liposomal amphotericin, 1 caspofungin, 1 itraconazole, and 1 voriconazole), and 2 patients received no antifungals (Table 1).

Measurement of Cyclophosphamide (CY) and Its Metabolites 

Blood samples were drawn from a central venous access catheter at end, 4, 8, 20 and 24 hour post end of each of the 2 CY infusions. A 5-mL sample of blood was collected at each of these time points and divided between 1 tube containing EDTA for CY and CEPM quantitation, and a second tube containing phenylhydrazine HCl to stabilize HCY [22]. All samples were stored at the bedside at 4°C until sample transport within 12 hours to the Pharmacokinetics Laboratory for quantitation of the plasma concentrations of CY, HCY, and CEPM by liquid chromatography and mass spectroscopy methods [22]. The exposure to CY and its metabolites was calculated by determining the AUCCY, AUCHCY, and AUCCEPM for time 0 to 48 hours, using noncompartmental analysis.

Evaluation of Clinical Outcomes 

Liver toxicity was defined by the presence of SOS, and its severity was recorded [15, 23]. Patients without evidence of liver disease were categorized as not having SOS, and patients in whom liver disease developed before day 20 after HCT that did not meet the criteria for SOS (but might result from, e.g., to acute graft-versus-host disease or cholangitis lenta) were categorized as having liver disease of unknown etiology, as previously described [23]. Liver toxicity was assessed without knowledge of the pharmacokinetic data. Death that occurred following progression or recurrence of the underlying hematologic disease was categorized as resulting from disease recurrence (relapse), regardless of the proximate cause; death in the absence of relapse was categorized as NRM. The time to last follow-up for overall survival is a median of 717 days (range: 300-1916) for those who received TBU/CY.

Statistical Methods 

Pharmacokinetic data in patients who received TBU/CY and CY/TBI were compared using the 2-sample t-test (for unadjusted comparisons) and linear regression (for age-adjusted comparisons). Patients were categorized as receiving fluconazole versus those who did not, to allow for comparison of pharmacokinetics between the 2 conditioning regimens. For patients conditioned with TBU/CY, logistic regression was used to assess the relationship between the exposure (i.e., AUC) to CY and its metabolites and the occurrence of SOS. Associations between the exposure to CY and its metabolites and the time-to-event outcomes relapse, overall mortality, and NRM (occurring by day 200 and to time of last follow-up) were assessed using Cox regression. Regression models were adjusted for type of donor (HLA identical sibling vs. others) and patient age at the time of HCT. AUC was modeled as a continuous linear variable in the regression models, and nonlinear terms were additionally examined to account for the possibility of such associations. Two-sided P-values from regression models were obtained from the Wald test, and no adjustments were made for multiple comparisons.

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Results 

Busulfan Pharmacokinetics 

After a 1 mg/kg dose of oral busulfan, the mean (±SD) plasma level was 976 ± 171 ng/mL with a 2.2-fold range (592-1312 ng/mL). The target Css range was achieved in 93% of patients. Specifically, among 69 patients with a target Css of 800-900 ng/mL, 64 achieved this target with a mean oral BU total dose of 14.6 (range: 9.8-22.3) mg/kg. The BU Css was 911-963 ng/mL, that is, outside of the target range, in 5 patients. All 5 CML patients with a target BU Css >900 ng/mL reached that target Css (range: 923-959 ng/mL), and the 1 patient whose target was 900-1000 ng/mL had a BU Css of 946 ng/mL. The fluctuations in BU dosing to achieve target Css are described in Table 2, based on whether the patient developed SOS.

Table 2. Relation of Busulfan Dose Adjustments to Achieve Target Css and the Development of SOS
SOS
No (N = 50)Yes (N = 17)
Dose 50.95±0.160.89±0.13
Dose 1(0.69-1.38)(0.67-1.23)
Dose 90.99±0.090.96±0.06
Dose 5(0.8-1.24)(0.8-1.03)

Busulfan dose 1 was 1 mg/kg for all patients; doses 5 and 9 were adjusted to achieve target Css.

Css indicates steady state plasma concentration; SOS, sinusoidal obstruction syndrome.

Comparison of CY Pharmacokinetics in Patients Who Received CY First in Order (i.e., CY followed by TBI) versus Second in Order, following TBU 

Peak plasma concentrations and AUCs of CY, HCY, and CEPM are summarized in Table 3. There was considerable interpatient variability in exposure (expressed as the AUC from 0 to 48 hours) to CY (8.7×), HCY (5.4×), and CEPM (8.1×). Patients who received TBU/CY were, on average, older than those who received CY/TBI (46.6 years vs. 35.3 years, P < .0001), and subsequent analyses comparing the AUCs of CY, HCY, and CEPM were adjusted for age using linear regression models. Patients receiving TBU/CY had a statistically significantly lower AUCCY and higher AUCHCY in comparison to those receiving CY without preceding exposure to BU and phenytoin (i.e., those conditioned with CY/TBI [15]). These differences were essentially unchanged after adjusting for age (Table 3). Patients who received TBU/CY also had higher AUCCEPM, but the difference was not statistically significant after adjusting for age (Table 3). The unadjusted AUCs are depicted in Figure 1.

Table 3. Comparison of Pharmacokineticsa of CY, HCY, and CEPM by Conditioning Regimen
CY/TBITBU/CYP-ValuebAge-Adjusted Difference,cP-Value
CY
Peak [CY]a, day 1364±52312±171<.000167,<.0001
Peak [CY], day 2358±54283±124<.000185,<.0001
AUCCY6134±12492563±1190<.00013887,<.0001
HCY
Peak [HCY], day 17±435±18<.0001−29,<.0001
Peak [HCY], day 214±736±13<.0001−23,<.0001
AUCHCY152±62290±98<.0001−134,<.0001
CEPM
Peak [CEPM], day 111±527±12<.0001−15,<.0001
Peak [CEPM], day 221±932±29<.0001−6,<.04
AUCCEPM416±186522±194<.0001−40,.15

AUC indicates area under the curve; CEPM, carboxyethylphosphoramide mustard; HCY, hydroxycyclophosphamide; TBI, total body irridiation; BU, busulfan; CY, cyclophosphamide.

aPeak concentrations (μM) are the highest concentration quantitated upon that day; AUC (μM·h) is from time 0 to 48 hours.

bTwo-sample t-test unadjusted for age.

cCY/TBI-TBU/CY, P-values adjusted for patient age at transplant.

  • View full-size image.
  • Figure 1. 

    Comparison of CY, HCY, and CEPM exposure by conditioning regimen. The AUC0-48hr in patients receiving CY/TBI (gray shading) and TBU/CY (no shading). Box designates 25th, 50th, and 75th percentile. Whiskers designate the extremes with one AUCCEPM of 1881 μM·h in the CY/TBI (gray shading) being excluded for easier presentation.

The difference in AUCs between the 2 conditioning regimens did not appear to be dependent on concomitant fluconazole use. There was a suggestion that the difference for AUCCY between CY/TBI and TBU/CY was dependent on concomitant fluconazole (P = .08). Patients conditioned with TBU/CY had a lower AUCCY than those who received CY/TBI, regardless of fluconazole use. In patients receiving concomitant fluconazole, the mean AUCCY for TBU/CY was 2894 ± 1173 μM·h and for CY/TBI was 6236 ± 1221 μM·h (P < .0001). In those not receiving fluconazole concomitant with CY, the mean AUCCY for TBU/CY was 1588 ± 517 μM·h and for CY/TBI was 5638 ± 1288 μMh (P < .0001). A test of interaction between conditioning regimen and concomitant fluconazole use yielded a P = .94 for AUCHCY and P = .61 for AUCCEPM, indicating that concomitant fluconazole use did not have a significant impact on the difference in AUCHCY and AUCCEPM between the 2 conditioning regimens.

The association between the AUCCY and its metabolites was also examined in an attempt to confirm our findings of conditioning regimen-dependent pharmacokinetics in a previously reported limited number of patients who had received BU/CY (N = 7) or CY/TBI (N = 7) [18]. In patients receiving CY/TBI, there was little correlation between AUCCY and AUCHCY (R2 = .034, top panel of Figure 2) or AUCCEPM (R2 = .005, bottom panel of Figure 2). Similarly, in patients receiving TBU/CY, there was little correlation between AUCCY and AUCHCY (R2 = .0004, top panel of Figure 2) or AUCCEPM (R2 = .02, bottom panel of Figure 2). The associations between average BU Css and the AUC of CY and its metabolites were weak (R2 = .08 for AUCCY, R2 = .02 for AUCHCY, and R2 = .0002 for AUCCEPM).

  • View full-size image.
  • Figure 2. 

    Relationship between AUC of CY and its metabolites in patients receiving preparative regimens of TBU/CY and CY/TBI. Top panel, AUCCY (x-axis) versus AUCHCY (y-axis); bottom panel, AUCCY (x-axis) versus AUCCEPM (y-axis).

In summary, the administration of i.v. CY after BU/phenytoin led to a marked reduction in the AUCCY and an increased AUC of both metabolites in comparison to CY administered first (i.e., CY followed by TBI). The AUC of the metabolites was not influenced by concomitant administration of fluconazole with CY and also could not be predicted by the exposure to CY or BU.

Pharmacodynamic Relationship between Clinical Outcomes and CY and Metabolite AUCs after TBU/CY 

The AUCs for CY, HCY, and CEPM were modeled as continuous linear variables with respect to each endpoint, as higher order terms for AUC did not statistically significantly improve the models containing only a linear term. Moderate to severe SOS was not statistically significantly related to AUCCY, AUCHCY, or AUCCEPM (Table 4). The relationships between AUCCY, AUCHCY, or AUCCEPM and relapse, NRM, or survival are summarized in Table 5. NRM at day 200 was related to AUCCY (p = .03), but the association was no longer significant at the time of last follow-up. Fifteen of the 75 TBU/CY conditioned patients (20%) died from nonrelapse causes, 5 from infections (3 with aspergillosis, 1 with encephalitis/ventriculitis, 1 with disseminated cytomegalovirus disease), 4 from pulmonary complications (2 adult respiratory distress syndrome; 1 pneumonia; 1 idiopathic pneumonia syndrome), 2 from multiorgan failure, 2 from graft-versus-host disease (GVHD), 1 from SOS, and 1 committed suicide. Further, AUCCY, AUCHCY, or AUCCEPM were not correlated with relapse or overall mortality.

Table 4. Frequency of Sinusoidal Liver Toxicity among Patients Conditioned with TBU/CY
SOS
No (N = 50)Yes (N = 17)P-Valueb
AUCCYa2450±12132680±1046.49
AUCHCY293±109287±67.82
AUCCEPM483±183550±162.18

AUC indicates area under the curve; CEPM, carboxyethylphosphoramide mustard; HCY, hydroxycyclophosphamide; BU, busulfan; CY, cyclophosphamide.

aAll AUCs expressed as AUC from time 0 to 48 hours in units of μM·h.

bTwo-sided p-values from regression models adjusted for age at time of HCT and type of donor. With exclusion of eight (11%), patients were classified as having liver disease of unknown etiology.

Table 5. Relationship between Exposure to CY and Its Metabolites and Clinical Outcomes among Patients Conditioned with TBU/CYa
Pharmacokinetic Parameters
Clinical OutcomeNbAUCCYAUCHCYAUCCEPM
Day 200 Nonrelapse mortality9HR = 0.36(P = .03)HR = 1.11(P = .71)HR = 1.01(P = .97)
Nonrelapse mortality15HR = 0.76(P = .31)HR = 0.82(P = .45)HR = 1.01(P = .96)
Relapse16HR = 0.81(P = .43)HR = 0.82(P = .49)HR = 0.88(P = .46)
Overall mortality26HR = 0.85(P = .41)HR = 0.76(P = .24)HR = 0.99(P = .91)

AUC indicates area under the curve; BU, busulfan; CY, cyclophosphamide.

aAUC modeled as continuous linear variable, with hazard ratios for AUCCEPM and AUCHCY representing increase in hazard ratio (HR) associated with increase in AUC of 100 μM·h. HR for AUCCY represents increase in hazard associated with increase in AUC of 1000 μM·h. HR adjusted for age at time of HCT and type of donor.

bNumber of events in cohort of 75 patients.

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Discussion 

The key findings of this study were the striking pharmacokinetic differences in CY and its metabolites between the myeloablative conditioning regimens TBU/CY and CY/TBI, and the apparent lack of a pharmacodynamic relationship between exposure to CY and its metabolites with clinical outcomes following the TBU/CY regimen. In the cohort of patients receiving TBU/CY, the AUCs of CY and HCY (but not CEPM) were profoundly affected by BU and the concomitantly administered phenytoin, compared to CY given first in order (Figure 1). In patients receiving TBU/CY, the CY metabolism showed large interpatient variability when CY dose (mg/kg), phenytoin dose (mg/kg), and BU Css (ng/mL) were held constant between patients (Figure 1). In comparison to CY/TBI patients in whom CY was administered first, TBU/CY patients had a decreased AUCCY and increased AUCHCY, whereas the difference was not statistically significant for AUCCEPM after adjusting for patient age. Our finding of age-dependent pharmacokinetics of CY is consistent with previous reports and was adjusted for within our statistical analysis [24, 25, 26, 27, 28, 29]. Fluconazole use did not appear to have an impact on the difference between conditioning groups for the AUCs of CY, HCY, and CEPM [21, 30, 31]. Our findings of altered CY pharmacokinetics with preceding BU/phenytoin were also consistent with previous reports, with the addition of data related to exposure to CEPM [18, 32].

Busulfan without phenytoin has been shown to inhibit CY metabolism [33]. The interaction between phenytoin and CY is complex in that competitive inhibition or induction (or both) could occur. Phenytoin is a substrate for CYP2C9, CYP2C19, and ABCC2 [34, 35]. Thus, phenytoin could competitively inhibit the metabolism of CY to HCY by CYP2C9 and CYP2C19 [36, 37] and the elimination of glutathionylcyclophosphamide by ABCC2 [38]. Phenytoin is also a well-recognized inducer of CYP2B6, 2C, and 3A, all of which catalyze HCY formation [34, 39]; phenytoin has not been reported to induce hepatic ABCC2 activity. In the data presented here, the inductive effects of phenytoin were evidenced by the decreased AUCCY and increased AUCHCY. Pretreatment with phenytoin increased AUCHCY by approximately 100%, which is consistent with other reports [18, 40]. Patients conditioned with CY/TBI had dramatic changes in the pharmacokinetics of CY, HCY, and CEPM between dose 1 and 2 because of CY autoinduction. Patients receiving TBU/CY showed minimal differences between the metabolite AUCs of dose 1 and dose 2, indicating that CY autoinduction was not a major factor in patients who received another inducer (i.e., phenytoin), presumably because the maximal metabolic rate was already obtained [41, 42]. In agreement with our previous data, there was only little correlation between the systemic exposure to CY (i.e., AUCCY) and its metabolites HCY and CEPM (Figure 2) [18]. Similarly, BU Css was not strongly associated with the AUCCY, AUCHCY, and AUCCEPM.

The lack of a statistically significant relationship between systemic exposure to CY and its metabolites and clinical outcome contrasts with our report that showed that AUCCEPM was strongly correlated with liver toxicity and mortality in patients conditioned with CY/TBI [15]. TBI is toxic to hepatic sinusoids, as demonstrated by the work of Geraci et al. [43] in a rat model and in our own clinical pharmacodynamic study [15]. The lack of a pharmacodynamic relationship with liver toxicity also contrasts with the observation that reducing the CY dose from 200 mg/kg to 120 mg/kg lowered liver toxicity in patients receiving BU/CY conditioning [44]. Liver toxicity was not observed after administration of single agent CY 150 mg/kg [45] or 200 mg/kg [46]; however, multiple case reports have been published of the liver toxicity in patients receiving single-agent CY [47, 48, 49]. Single-agent therapy with oral BU (1 or 1.25 mg/kg every 6 hours for 16 doses) led to bilirubin greater than 2 mg/dL in all 6 patients; however, 1 patient had no evidence of drug toxicity upon autopsy and another had chronic active hepatitis [50]. As a pharmacodynamic relationship was not apparent, other risk factors for SOS and NRM are likely contributing to the toxicity of this TBU/CY preparative regimen.

The available data suggest that the concentration effect (i.e., the pharmacodynamic) relationship of CY and its metabolites is dependent upon the conditioning regimen [15, 51, 52, 53]. This trend has been observed specifically with BU in that BU exposure was related to liver toxicity in BU/CY, but not BU/melphalan or BU/thiotepa regimens [2, 3, 4]. Also, the pharmacodynamic relationship of BU and liver toxicity in adults differ based on disease, such that higher BU Css is associated with a higher risk of liver toxicity in patients with diseases other than CML in the chronic phase [2, 3, 4, 13, 54, 55, 56, 57, 58].

Nevertheless, the TBU/CY regimen used here resulted in a 20% NRM rate at day 200 post-HCT, which raised the question as to whether regimen-related toxicity from TBU/CY could be reduced by modification of the regimen. In the BU/CY regimen, i.v. busulfan has been reported to result in lower rates of SOS than the oral BU, when both were given on the basis of body weight and not dose adjusted to achieve target Css [9, 10]. In a cohort of patients receiving BU without phenytoin followed by CY, Hassan et al. [33] observed a higher AUCCY, lower AUCHCY, and higher incidence of SOS with a 7-15 hour time interval between BU and CY administration in comparison to a 24-48 hour time interval between the last BU dose and CY, suggesting that a prolonged time interval between administration of BU and CY may be beneficial. Switching the administration order to CY followed by BU (i.e., CY/BU) may decrease liver toxicity and, thus reduce NRM, although its impact upon efficacy has not been evaluated. Preclinical data in mice suggest that a CY/BU conditioning regimen leads to more rapid engraftment of donor cells and lower plasma cytokine and serum hepatic enzyme levels than observed with BU/CY [59]. In addition, the incidence of SOS was significantly lower when BU was administered second, not first, in children receiving myeloablative conditioning regimens with 3 alkylating agents [60]. Another strategy to reduce regimen-related toxicity may be to abandon CY in favor of fludarabine, that is, to use a BU/fludarabine regimen. The BU/fludarabine regimens studied thus far appear to have lower rates of regimen-related toxicity than BU/CY regimens, although randomized trials are needed to compare not only efficacy, but also long-term toxicity of these 2 conditioning regimens [61, 62, 63].

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Acknowledgments 

This study was supported by grants from the National Institutes of Health (CA15704 and CA18029). We thank the medical and nursing staffs, along with the patients and the patients’ caregivers for their support in the completion of this study.

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PII: S1083-8791(07)00236-4

doi:10.1016/j.bbmt.2007.03.012

Biology of Blood and Marrow Transplantation
Volume 13, Issue 7 , Pages 853-862, July 2007

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