The lower the MAPE value, the better the LSS. A sampling strategy was considered to display a good predictive performance when the 95% confidence interval around the MAPE was less than 20% of the reference Bu AUC0-6h values (33). Bland-Altman plots were applied to assess the agreement between the predicted and the actual AUC0-6h, and the difference was expressed as a mean ± 1.96 SD.
2.7 Definition of Clinical Outcomes
The definition of engraftment was absolute neutrophil count (ANC) ≥ 0.5 × 109 per liter for the first three days in a row and platelet count (PLT) ≥ 20 × 109 per liter for the first one week after HSCT (10, 16). Primary engraftment failure was defined as failing to reach an ANC of 0.5 × 109 /L within 30 days after HSCT (16). In brief, the diagnosis of SOS was according to the modified Seattle criteria (34). The Mount Sinai Acute GVHD International Consortium (MAGIC) was taken as the diagnostic criterion for aGVHD (35).
2.8 Statistical Analysis
Continuous data, like pharmacokinetics parameters, was compared using a two-tailed t-test, whereas time-to-event data was compared by the log-rank test. Univariate analysis was performed with GraphPad Prism (GraphPad Software, San Diego, CA). In this study, p values < 0.05 are considered as statistical significance, andp values < 0.1 were deemed as trends.
Results
3.1 Population characteristics
From March 2019 to April 2020, 76 patients underwent allogeneic HSCT and received IV busulfan. The GSTA1 genotypes for four patients andGSTM1 genotypes for six patients were missing because of samples being unavailable or detection failure. GSTA1-52C and Tdefined haplotype *A and *B , respectively. In relation toGSTA1-52T/C and -69A/G SNPs, a higher percentage ofCC/GG (78.08%) versus TT/AA (1.37%) and CT/GA(20.55%) was found in our model-building patient cohort. There was only one patient with GSTA1 *B*B . Thus, 69 patients were included in our final PPK analysis. Table 1 summarizes characteristics of patients including demographics, primary diseases, donor types and conditioning regimens. Supporting Information Table 3 further elaborates patients’ diagnoses. The genotyping results are shown inTable 2 .
3.2 PPK model
A total of 398 blood samples were collected for the PPK analysis.Supporting Information Figure 1 plotted concentration–time profiles of Bu. A one-compartment model with first-order elimination was suitable to describe the profiles of busulfan pharmacokinetics, and the exponential model was feasible to estimate the inter-individual variability. Neither proportional error model nor additive error model could perform well, while a combined proportional and additive residual error model provided an adequate fit (Δ-2LL > 20, p < 0.001).
In the forward model building step, BSA, AST, types of primary diseases and GSTA1 genotypes declined the value of -2LL by more than 3.84 (p < 0.05) at each addition. The GSTA1 mutation, BSA and the level of AST significantly affected the clearance (CL) of busulfan. Meanwhile, the volume (V) of busulfan was influenced by BSA and types of primary diseases, while the GSTM1 genotypes had no significant effect on PK parameters.
During backward elimination, the value of -2LL increased significantly (Δ-2LL >6.63, p < 0.01) when respectively removing the GSTA1 mutation, BSA and the level of AST out of the model. Thus, the three covariates mentioned above could remain in the final model. Types of primary diseases were eliminated in the final model because it failed to significantly increase the -2LL value (Δ-2LL = 6.333, p > 0.01). Table 3 presented the detailed PK parameters of the base and final model. The CL and V of busulfan were affected by the GSTA1 genotypes, BSA and AST as illustrated by the following Equation 5 and 6 :