Biology of Blood and Marrow Transplantation 5:316–321 (1999) © 1999 American Society for Blood and Marrow Transplantation
Myeloablation by intravenous busulfan and hematopoietic reconstitution with autologous marrow in a canine model H. Joachim Deeg,1 Ulrich S. Schuler,2 Howard Shulman,1 Michael Ehrsam,2 Ulf Renner,2 Cong Yu,1 Rainer Storb,1 Gerhard Ehninger2 1
Clinical Research Division, Fred Hutchinson Cancer Research Center, and University of Washington School of Medicine, Seattle, Washington; 2Medizinische Klinik und Poliklinik I, Universitätsklinikum, Dresden, Germany Offprint requests: H. Joachim Deeg, MD, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., D1-100, P.O. Box 19024, Seattle, WA 98109-1024; e-mail:
[email protected] (Received 24 March 1999; accepted 20 May 1999)
ABSTRACT We have previously described pharmacokinetic studies with a dimethylsulfoxide-based intravenous busulfan preparation in a canine model and in preliminary clinical trials. Using the same intravenous busulfan preparation, we carried out a dose escalation study to determine a marrow-ablative dose and to test the ability of autologous marrow to reconstitute hematopoiesis in dogs so treated. Busulfan was given intravenously at doses of 3.75 to 40 mg/kg. Marrow ablation was achieved at 20 mg/kg given either as a single dose or in four daily increments of 5 mg/kg each. There was a relative sparing of lymphocytes. A busulfan dose of 40 mg/kg resulted in severe central nervous system toxicity. Otherwise, nonhematopoietic toxicity was minimal and restricted to mild hepatic abnormalities. Four dogs were given busulfan at 20 mg/kg followed 30 hours later by infusion of autologous marrow, and all showed prompt and complete hematopoietic reconstitution. The area under the curve (AUC) determined by busulfan concentration in plasma over time was dose dependent, ranging from 12 to 100 mg · h/mL for busulfan doses of 3.75–20 mg/kg. There was a suggestion that the plasma half-life increased at the highest busulfan doses used. Intravenous administration of busulfan circumvented differences in bioavailability; nevertheless, considerable variations in the pharmacokinetic parameters were observed between individual animals. Thus, intravenous busulfan can be given safely and is effective in ablating hematopoiesis. However, factors other than absorption influence the AUC, and individualization of dosing may be required even with intravenous administration of the drug.
KEY WORDS Autologous stem cell transplantation
•
Canine model
INTRODUCTION Oral high-dose busulfan is used widely as a myeloablative and antileukemic treatment in preparation for bone m a rrow or peripheral blood stem cell transplantation (hematopoietic stem cell transplantation [HSCT]) [1–3]. In randomized studies comparing busulfan with irradiationcontaining regimens, comparable overall efficacy was shown in all but one study in patients with acute myelogenous leukemia [4]. The use of busulfan is not without problems, however [5–10]. Highly variable busulfan concentration Supported by grant W 14/90/EhI from the Deutsche Krebshilfe e.V. and grants HL36444 and CA15704 from the National Institutes of Health, Bethesda, MD.
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• Intravenous busulfan
• Myeloablative dose
profiles have been observed, presumably caused by variable gastrointestinal absorption [11,12]. The incidence of hepatic veno-occlusive disease (VOD), an often life-threatening regimen-related toxicity, appears to increase with exposure to higher busulfan levels [5,9,10,13,14]. At the same time, reduced busulfan concentrations might be associated with increased risks of graft rejection and recurrence of leukemia [10]. For this reason, studies performed with exposure-guided dose adjustment employing area under the curve (AUC) analysis have aimed at achieving safer or more efficacious plasma busulfan concentrations [15–18]. One strategy that may circumvent pharmacokinetic problems related to variations in bioavailability is the use of an intravenous formulation of busulfan.
We previously reported on the development of such a p reparation and showed in a canine model that single intravenous doses of 1 mg/kg busulfan resulted in peak plasma levels of 730–1000 ng/mL and AUC values of 75–146 ng · h/kg · mL [19]. The same preparation has also been tested in a clinical pilot study [20]. Different intravenous preparations of busulfan—using, for example, solutions of DMSO, ethanol and polyethylene glycol (PEG) [21], or dimethylacetamide and PEG [22,23]—have been tested by other investigators. However, no systematic dose-response study of intravenous busulfan in regard to myelosuppression has been presented. Here we carried out a dose escalation study in dogs to determine a myeloablative dose of the intravenous busulfan preparation and the ability of autologous marrow to reconstitute hematopoiesis in dogs so treated.
MATERIALS AND METHODS Dogs Sixteen beagles, 8 to 20 months old and weighing 7.8–20 kg (median 11), were housed and provided with commercial chow and drinking water ad libitum as described [24]. Kennel facilities and animal care were in accordance with the guidelines stated by the National Academy of Sciences. The experimental protocol was approved by the Institutional Animal Care and Users Committee of the Fred Hutchinson Cancer Research Center. For 12 hours before each experiment, dogs received no food but had free access to drinking water. All dogs had peripheral blood sampled for busulfan pharm a c o k i n e t i c analysis as described below. Autologous marrow transplants were carried out as described below. Busulfan was administered intravenously at total doses of 3.75, 7.5, 10, 15, 17.5, 20, or 40 mg/kg (Table 1). In 15 dogs, the dose was given as a single injection; one dog (D959) received the total dose of 20 mg/kg in four increments (5 mg/kg/day for four consecutive days). Drug pr eparation The intravenous formulation was prepared immediately before administration as described [19,20,25], and no PEG was added. Briefly, busulfan powder (Aldrich Chemical, Steinheim, Germany) was dissolved in 1 mL dimethylsulfoxide (DMSO), the amount required for treatment was filtered (0.2 mm PTFE filter; Lida Manufacturing, Kenosha, WI) into 50 mL 0.9% sodium chloride, the concentration was verified, and the solution was administered over 2 minutes via an intravenous cannula. The measurements of busulfan concentration in aliquots showed no loss during the preparation steps. Degradation in the solution if kept at 20°C occurred at a rate of 5% per hour (determined over 18–24 hours). Blood samples Blood samples were obtained before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 24, and 48 hours after dosing [19]. In the single dog (D959) given 5 mg/kg for four doses, blood was sampled only for 24 hours, that is, until administration of the second busulfan dose. The dog (E153) given 40 mg/kg intravenously as a single dose developed a generalized convulsion upon completion of the injection [7], had a respiratory arrest, and died. Thus, no postinjection blood samples
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Table 1. Pharmacokinetic parameters in dogs given intravenous busulfan Dog E180 E959* D979 D943 E059 E041 E039 D942 E042 E040 D983 E285 E286 E426 E455
Body weight (kg) 10.4 12.2 8.3 12.9 7.8 11.4 11.1 19.4 11.4 12.4 11 9.3 10.4 10 14.2
Dose (mg/kg) 3.75 5.0 7.5 10 15 15 15 17.5 17.5 17.5 20 20 20 20 20
t
1/2
(h)
1.54 1.54 0.8 0.97 0.83 1.38 1.04 1.34 1.12 0.93 1.09 2.46 2.55 1.84 2
AUC ( mg · h/mL) 19 13 12 20 23 49 31 57 32 41 48 86 100 79 73
Clearance (mL/h) 191.3 370.4 604.8 483.1 632.9 305.5 471.7 305.4 541.8 419.7 409.0 232.6 198.8 251.2 270.6
*Dog E959 received 5 mg/kg/day on four consecutive days for a total of 20 mg/kg; by necessity, pharmacokinetic studies for the 5 mg/kg dose could be determined only for 24 hours after the initial dose.
were available. Blood samples were immediately placed on ice and centrifuged at 4000g at 4°C. Plasma was separated and stored at –20°C until analysis. Analysis Busulfan in plasma was assayed using a pre v i o u s l y de scrib ed hig h-perf o rm ance l iq uid c hro m a t o g r a p h y (HPLC) method with minor modifications [25]. In brief, solid-phase extraction on Bond Elut C8 cartridges was used for sample cleanup. In a precolumn derivatization, the extracted busulfan was converted into 1,4-diiodobutane. The separation of 1,4-diidobutane was achieved with an isocratic HPLC system using a guard column of 5 mm LiChrosorb CN (E. Merck, Darmstadt, Germany) and a Gromsil 100CNN analytical column (M. Grom, Herrenberg, Germany). The solvent system was water-acetonitrile (78:22, vol/vol) at a flow rate of 1 mL/min. A postcolumn photochemical derivatization with a Teflon reactor (inner diameter 0.8 mm; length 6 m; MSD Metron GmbH, Aschheim, Germany, and Gynkotek, Germering, Germany) enabled the detection of the busulfan derivative at 226 nm with a retention time of 16.5 minutes. This method has a high selectivity. The coefficient of variation at a concentration of 250 ng/mL was 2.7%, and the detection limit for busulfan in plasma was 20 ng/mL. Pharmacokinetic parameters were determined by use of the TOPFIT program, and AUC was calculated by use of the trapezoidal rule. Assessment of my eloablation Peripheral blood cell counts were obtained before and at daily intervals after completion of busulfan administration. Bone marrow cellularity was determined at autopsy. Largely based on earlier studies with dimethylbusulfan [26] and more recent work using various conditioning regimens [24], a decline in neutrophils to ,10/mL combined with a platelet
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count of ,103/mL and marrow cellularity of ,5% of normal were the criteria used for myeloablation. Transplantation Four dogs had their autologous marrow aspirated under general anesthesia and cry o p re s e rved as described [24]. Dogs were allowed to recover for 10–14 days after the marrow harvest, were treated with busulfan (20 mg/kg intravenously), and had the thawed marrow infused intravenously 30 hours after completion of the busulfan infusion. The dose of mononuclear cells administered was 3.3–5.13108/kg (median 4.53108/kg). Supportiv e care Dogs were maintained on oral fluid and food intake as tolerated. Complete blood counts were obtained daily beginning with the day of busulfan administration and continued until hematopoietic recovery or death with aplasia. No seizure prophylaxis was given. When the absolute neut rophil count (ANC) declined to ,131 09/L, dogs were started on oral nonabsorbable and systemic antibiotics as described [24]. Dogs were checked and weighed daily, and the findings were recorded in individual records. All dogs that died spontaneously or were killed had complete autopsies (including marrow examination) performed as described [24].
RESULTS Dose escalation and my elosuppression Figure 1 summarizes hematologic results of the dose escalation study. Dogs experienced a busulfan dose–dependent decline in peripheral blood cell counts. At doses of 3.75–15 mg/kg, all dogs recovered hematopoietic function and survived. One of three dogs given 17.5 mg/kg and both dogs given 20 mg/kg (including the dog given four increments of 5 mg/kg) died with marrow aplasia.
White blood cell (WBC) count With increasing doses of busulfan there was a progressively steeper decline in WBC, with progressively lower nadirs reached between days 15 and 18 after busulfan injection. Even at the highest dose of busulfan (20 mg/kg), the WBC nadir did not fall below 13103/mL; remaining cells were exclusively lymphocytes and monocytes. Neutrophils The decline in neutrophils in general paralleled that in WBC. However, the higher the busulfan dose, the earlier the nadir was reached (day 18 for 5 mg/kg; day 13 for 20 mg/kg). At a dose of 20 mg/kg busulfan, the mean neutrophil count declined to ,10/mL. Platelets Similarly, the platelet nadir (reached at 16–33 days) was busulfan dose–dependent, although the kinetics of recovery were somewhat obscured due to platelet transfusion support given to prevent hemorrhage. At busulfan doses of 15–20 mg/kg, the platelet nadirs were below 103/mL. On the basis of these findings (and the seizure-related death at a dose of 40 mg/kg) 20 mg/kg was considered the marrow-ablative dose that was not associated with clinically apparent non-marrow toxicity. Hematopoietic r escue Four dogs (E285, E286, E426, E455) were given 20 mg/kg busulfan intravenously and, after a 30-hour interval, were infused with previously cryopreserved autologous marrow. All four survived. Results are illustrated in Figure 2. The nadirs for WBC, neutrophils, and platelets were less profound than in dogs not given marrow rescue, due to the beginning of reconstitution by infused marrow cells. Decline and recovery of WBC and neutrophils were quite homogeneous for all four dogs, while in one dog (E286), platelet recovery was somewhat protracted ($105/mL on day 37).
Figure 1. Peripheral blood cell counts in dogs given intravenous busulfan at doses of 3.75–20 mg/kg and no marrow rescue Shown are the mean values of granulocytes (A) and platelets (B) by day after busulfan injections in four groups of dogs. Group 1 (m) includes dogs E180 (3.75 mg), D979 (7.5 mg), and D943 (10 mg); group 2 (★) includes dogs E039, E041, and E059 (each given 15 mg/kg); group 3 (d) includes dogs D942, E040, and E042 (each given 17.5 mg/kg); group 4 (j) includes dogs D959 and D983 (each given 20 mg/kg).
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Figure 2. Peripheral blood cell counts in four dogs Dogs E285, E286, E426, and E455 were given 20 mg/kg busulfan followed 30 hours later by the infusion of autologous marrow cells. Data shown represent values in individual dogs.
Busulfan pharmacokinetics Pharmacokinetic results in the 15 evaluable dogs are summarized in Table 1. After doses of 3.75–20 mg/kg, peak plasma levels of 7279 to 25,582 ng/mL were measured. The AUC ranged from 12 to 100 mg · h/mL. Elimination halflives were 0.8–2.55 hours. The t1/2 increased with the dose of busulfan given. The AUC also increased, as expected, with increasing busulfan doses. However, there was no tight correlation with AUC for a given dose level, with values varying by as much as 106% (Table 1). Variations in plasma clearance were even broader, ranging from 191.3 to 632.9 mL/hour (331%), and it is mostly on this basis that the AUCs varied. These data show, therefore, that variability in plasma levels is related not only to differences in bioavailability (after oral administration) but also to differences in drug elimination [13,27]. Toxicity and autopsy findings The tolerability of intravenous busulfan administration at doses of 3.75 to 20 mg/kg was excellent. One dog given 40 mg/kg died with neurotoxicity (convulsion). The major toxicity was, as intended, marrow suppression with relative sparing of lymphocytes. In dogs E059 and E040, given 15 and 17.5 mg/kg of busulfan, respectively, liver sections at autopsy showed focal congestion and subendothelial edema of central veins which, in E040, was associated with mild hepatocyte destruction in zone 3 and replacement of degenerative hepatocytes by dilated sinusoids resembling peliosis. In dog D983, given 20 mg/kg of busulfan and killed on day 25, the thymus failed to show clear cortico/medullary distinction. All dogs that died with marrow aplasia showed intestinal mucosal hemorrhage.
DISCUSSION Busulfan, a stem cell–toxic alkylating agent, is used extensively to condition patients for autologous and allogeneic HSCT [1–4,8,10,20]. Currently, the standard admin-
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istration of busulfan is in the form of an oral preparation, and major intra- and interpatient fluctuations in bioavailability have been reported [11,12]. In children, generally lower plasma levels than in adults are achieved after the oral administration of identical per-kilogram busulfan doses [10–13,27]. Furthermore, nausea and vomiting associated with oral busulfan tend to interfere with timely administration of the prescribed doses. Clinical toxicity appears to correlate with busulfan plasma levels [9,10,15], and re c e n t results indicate that sustained engraftment of donor cells and, at least in patients with chronic myelogenous leukemia, p revention of disease re c u rrence after transplantation depend on achieving effective drug plasma levels [2,10]. In an attempt to assure more consistent bioavailability, busulfan preparations for intravenous administration have been developed [12,19–23]. While several of these studies used PEG-containing solutions, we dissolved busulfan in DMSO and normal saline as used previously in pharmacokinetic studies in a canine model [19]. In the present study, we used the same model to define a marrow-ablative dose of intravenous busulfan and test the ability of autologous marrow cells to rescue dogs given myeloablative doses of intravenous busulfan. A busulfan dose of 20 mg/kg, given either as a single intravenous infusion or in increments of 5 mg/kg/day for four consecutive days, consistently resulted in marrow ablation with severe neutropenia and thrombocytopenia; there was only a slight decrease in lymphocyte counts, quite similar to earlier observations in dogs treated with dimethylbusulfan [28]. Nonhematologic toxicity was minimal at busulfan doses of 3.75–20 mg/kg and was limited to minor histologic alterations in the liver. Neurotoxicity in the form of convulsion and death in a dog given 40 mg/kg busulfan intravenously was not unexpected, considering the wellestablished effect of busulfan on the seizure threshold [7]. Pharmacokinetic data following intravenous administration of busulfan were more complex than anticipated. It appeared that at the highest dose level studied (20 mg/kg),
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plasma half-life may have been increased. Such a phenomenon might be related to saturation of a distribution or elimination process. The AUC increased progressively with the dose of busulfan administered. For any given dose level, however, there was considerable variation between individual animals. In the dose range where comparable data on human patients are available, AUCs in dogs were somewhat lower than would have been expected in humans, consistent with a higher clearance in dogs [19]. To achieve AUCs comparable to those obtained in humans, 2.5- to 4-fold higher busulfan doses were required in dogs. Extrapolation of the canine data to patients suggests that intravenous doses up to 4–5 mg/kg will be safe even if given as a bolus. Single oral doses of 4 mg/kg have been administered to patients without undue toxicity [27]. If the intravenous preparation is given over 2–4 hours rather than as a bolus, a plasma concentration profile similar to that obtained with oral dosing is expected. Such a prolonged infusion would also be associated with a lower peak level and, presumably, would be well tolerated. The present data show that, in addition to differences in absorption and bioavailability, important differences exist in the metabolism and elimination of busulfan dependent on the route of administration. It is likely, therefore, that even with intravenous administration of busulfan, monitoring of plasma levels and dose adjustment will be required to consistently reach a desired target concentration [11,13,15,16]. Nevertheless, these results may serve as a guide for the clinical use of intravenous busulfan, in regard to both dose requirements and dosing schedules. Hematopoietic reconstitution in four dogs given autologous marrow infusions 30 hours after busulfan administration was uneventful, an indication that the drug was cleared and the homing mechanism of hematopoietic stem cells and marrow microenvironment were intact. The present study was not designed to distinguish (for example, by gene marking) between recovery of hematopoiesis from infused vs. residual endogenous cells. However, the kinetics of granulocyte and platelet recovery clearly indicated that infused cells were essential to recovery. In comparison to dogs that were not rescued by marrow infusion, the blood cell nadirs reached were higher, due to beginning repopulation from the infused cells, and recovery was comparable to that seen in dogs rescued after dimethylbusulfan [28] or lethal doses of total-body irradiation [24,29]. The experiments described here did not investigate the immunosuppressive effects of intravenous busulfan. Similarly, it remains to be determined whether the present approach has the same antileukemic activity as the oral administration of busulfan. However, the fact that marrow ablation was achievable with a single intravenous dose is of interest and might render such an approach attractive. For example, cryopreservation of autologous marrow would not be necessary, since cells can easily be stored in the refrigerator for the required time interval. Whether it would be preferable to use other vehicles for the intravenous busulfan that provide different drug stabilities remains to be determined [21–23]. Thus, observations in this large animal model show that an intravenous preparation of DMSO-busulfan can be administered safely at doses sufficient to achieve marrow ablation. Hematopoietic reconstitution with autologous transplanted marrow cells is prompt and complete. Howev-
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er, while intravenous administration of busulfan circumvents p roblems associated with variable absorption after oral administration, it does not prevent individual variability related to drug elimination and other processes.
ACKNOWLEDGMENTS We thank Harriet Childs and Bonnie Larson for typing the manuscript and the technicians in the Canine Shared Facility for excellent care of the dogs included in this study. We also thank John Slattery, PhD, for reading the manuscript and offering his comments, and Gary Schoch for generating the figures.
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