Simulation of the of CD33-drug interaction dynamics in patients.

<p>The mechanism-based PK model (4) was used to simulate dynamics of free, cell surface-bound and intracellular drug levels during and after 2-hour intravenous infusion. Model parameter values were set at population means (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024265#pone-0024265-t001" target="_blank">Table 1</a>). The time series of each variable is depicted for short (<b>A</b>) and long (<b>B</b>) time period.</p

Estimation of the model parameters from in vitro experiments.

<p>Model parameters were estimated by fitting model equations to the data of in vitro interaction of GO with AML193 cells. AML193 cells were exposed continuously to either saturating GO concentration (5 µg/ml) for 1, 3, and 6 hours, or to non-saturating GO concentrations (0.1 to 0.5 µg/ml) for 15 minutes. In saturating experiments measured amounts of total (<b>A</b>) and bound (<b>B</b>) CD33 molecules per cell were fitted by equation (3); CD33 production rate (R_p), free (k_e) and bound CD33 internalization rates were estimated. In non-saturating experiments measured amounts of total (<b>C</b>) and bound (<b>D</b>) CD33 molecules per cell were fitted by equations (1); estimated parameters were drug-CD33 association (k_b) and dissociation (k_u) rates (R_p, k_e and k_i were set to their values obtained from the saturating experiments). Fitting was performed to the results of single representative experiments. </p

Effect of blast burden on intracellular exposure to GO.

<p>Parameter sensitivity analysis for CD33 production rate (<b>A</b>) and drug dose (<b>B</b>) was performed for various values of the initial blast burden (total number of blasts in the body). I-AUC and CD33 production rate are expressed in relative units as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024265#pone-0024265-g004" target="_blank">Figure 4</a>.</p

Lack of correlation between calculated intracellular exposure to GO with AUC of the drug in blood and CD33 expression level.

<p>The mechanism-based PK model (4) was used to simulate dynamics of free, cell surface-bound and intracellular drug levels during and after 2-hour intravenous infusion. (<b>A</b>) Correlation between blood vs intracellular AUC plot obtained by simulating the mechanism-based PK model and using wide range of model parameters (N₀, Dose, k_b, R_p, k_e, k_i set to tenth, one and ten times their average values). (<b>B</b>) Baseline CD33 antigen expression level vs. calculated intracellular AUC. Each point represents calculation for an individual AML patient. Variable units: AUC – ng/ml*day, I-AUC – molecules/cell*day, CD33 expression level – molecules/cell.</p

Parameter sensitivity analysis.

<p>Intracellular AUC of GO was calculated for different parameter sets. Each time only one parameter was varied, including CD33 production rate (<b>A</b>), association rate (<b>B</b>), initial blast burden (<b>C</b>), internalization rate (<b>D</b>), efflux rate (<b>E</b>), and total drug dose (<b>F</b>). All the other parameters were kept constant at their mean values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024265#pone-0024265-t001" target="_blank">Table 1</a>). I-AUC values were normalized to the value obtained for the set of mean parameter values. Parameter values are expressed in relative units after normalization to their mean values.</p

Estimates of the model parameters.

<p>*results of fitting clinical data are reported as mean, and inter-patient% CV (coefficient of variation)</p><p>for those parameters for which estimation in individual patients was possible; NM – not measured;</p><p>NA - not applicable; GO – Gemtuzumab Ozogamicin.</p