Mini Test Dose of Intravenous Busulfan (Busulfex®) in Allogeneic Non-Myeloablative Stem Cell Transplantation, Followed by Liquid Chromatography Tandem-Mass Spectrometry (2012)

Busulfan (Bu) has been used for almost 3 decades in the conditioning of hematopoietic stem cell transplant. Bu has a very diverse gastrointestinal absorption which in association with its emetic effect in high doses makes inter- and intra-individual oral bioavailability and thus systemic exposure very variable and difficult to predict (1, 2, 3). If the systemic exposure is below the therapeutic window there is an increased risk of lack of engraftment (4) and, in chronic myelogenous leukemia (CML) patients, disease relapse (5), while overexposure results in an increased incidence of high-dose related toxicities like hepatic veno-occlusive disease (6, 7). It is important to note that “busulfan concentration-response relationships are regimen-, age- and disease-dependent”, as emphasized by McCune et al (8). Therapeutic dose monitoring (TDM) gained an important role in the setting of oral Bu dose as it became clear that the level of systemic exposure influenced the toxicity, engraftment and relapse rates. To give an idea of the importance of TDM, a test dose prior to starting the conditioning regimen with home-administered oral Bu in 153 patients evidenced that as many as 68% of them would have drug exposures out of the therapeutic window (900–1,500 μM·min) with the standard dose of 1mg/kg of ideal body weight every 6 hours (9).

An intravenous (i.v.) formulation was developed to overcome these issues and avoid the utilization of TDM, However, in a series of 6 studies using intravenous Bu in adults, 72 to 91% of the patients achieved the target AUC without dose changes, while 10 to 82% of children achieved it in a series of 5 studies with the intravenous drug (10). Clearly, a need for TDM even when i.v. is used still persists. Many different strategies using single or repeated sampling in different points of the therapy were proposed to improve TDM results (10). However, since TDM can only be useful if Bu determinations are timely delivered and many transplant centers do not have a facility to perform Bu determination, new approaches using pharmacokinetic analysis of a sub-therapeutic test dose administered a few days earlier to predict the ideal dose were proposed. In one such study, 7 patients received a test dose of 0.8 mg/kg, followed by 0.8 mg/kg/dose every 6 hours for 15 doses (11). Bu concentrations were obtained and mean drug clearance was determined after the test, first and 13th doses. The utilization of a test dose demonstrated bioequivalence ( 20% variability). However, when the same 0.8 mg/kg test dose assay was compared to the intravenous Bu in continuous infusion for 90 hours in other 7 patients, they showed similar clearances (12). The next step was to examine the ability of the test dose clearance to base the calculation of the 90 hour continuous infusion dose (13). All 4 patients tested had AUCs within the desired range and no adjustments were required after 18 hours. More recently, Beri et al. (14) used a 0.8 mg/kg i.v. test dose (1/4 of the standard daily dose). Without the test dose, 23% of the 17 patients would have fallen below and 12% above the therapeutic range. With the test dose, no patients had a sub-therapeutic AUC and only 12% had an AUC above the therapeutic window.

In this work, in order to further reduce the unnecessary exposure and adverse effects of the test dose process, we investigated bioequivalence of a very low sub-therapeutic test dose (1/10 of the standard daily dose) of i.v. Bu (Busulfex®). For this purpose we developed a sensitive and fast liquid chromatography tandem-mass spectrometry (LC-MS/MS) method which allowed us to measure Bu in a wide concentration range required in the study.