BEVACIZUMAB EFFECT ON TOPOTECAN PHARMACOKINETICS ...

2.2.6 High-performance liquid chromatography analysis for PK studies
Total TPT in plasma and tumor ECF were measured as previously described [165,
180]. 20 μL plasma aliquots were added to 80 μL cold methanol (−30°C). Samples were
vortex mixed vigorously and centrifuged at 10,000 rpm for 2 minutes. TPT in the plasma
methanolic supernatants and tumor ECF were converted to total TPT by adding five parts
methanolic supernatant or tumor ECF to one part 20% phosphoric acid and injected into
HPLC with fluorescence detection. The unbound plasma TPT concentration was
calculated on the basis of a previous study [178], showing a 30.1% unbound fraction in
CB-17 SCID mice. All TPT concentrations reported in this study were the total unbound
TPT concentration.
2.2.7 Pharmacokinetic model evaluation
Different PK models were considered to describe the TPT concentrations in
plasma and tumor ECF: (1) a three compartmental PK model previously used in our lab
(Figure 3) [162, 165, 169]; (2) we also evaluated other multi-compartmental models
during the data analysis. The final modified multi-compartmental PK model is shown in
Figure 4.
The modified multi-compartmental PK model significantly improved the
estimation of PK parameters describing TPT in tumor compartment compared to the
conventional model. This general model can be applied for drug administration via orally,
IP or IV bolus. The model has an absorption compartment, a central and a peripheral
compartment, and a tumor compartment. Additionally, two transient compartments
between the plasma and tumor compartments were incorporated to account for the delay
of drug transport from plasma to tumor compartment. The differential equations are
shown in Equations 2-7. Model parameters estimated for intravenous dosing included
elimination rate constants (Ke is systemic elimination rate constant; Kcp, Kpc and Kct
are intercompartment rate constants; Kte is elimination rate constant for TPT leaving
tumor compartment) and the volume of distribution in central compartment (Vc) and
tumor compartment (Vt). The secondary parameters were determined: CLin = Kct × Vc
and CLout = Kte × Vt. The independent rate constants between the transient
compartments were not identifiable and were set to the elimination rate constant Kct. All
volume units are described in liters/m 2 and all elimination rates are described in hour -1 .
Eq. 2
Eq. 3
Eq. 4
Eq. 5
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