2.13. Pharmacokinetics and subcutaneous bioavailability of CA192

To study the pharmacokinetic profile of the CA192 carrier, Rho-CA192 (200 μM CA192 concentration; 104% labeling efficiency, 150 μL injection volume/35 g BW) was injected into 12-week male BALB/c mice intravenously (IV) or subcutaneously (SC) for a total dose of 30 nanomoles or 857 nanomoles / kg BW. 20 μL of blood was collected by tail nick at time points from 5 min to 72 hr. The collected blood was immediately added to 80 μL of heparinized PBS at a heparin concentration of 1000 U/ml. Red blood cells were removed by centrifugation at 16,100 x g for 10 min and diluted plasma was collected. CA192 concentration in plasma was calculated based on the fluorescence intensity measured by a SpectraMax iD3 Multi-Mode Microplate Reader (Excitation/Emission: 540/580 nm).

Both non-compartmental and compartmental methods were applied to analyze the PK profiles of CA192 after IV or SC administration. Non-compartmental analysis was primarily based on the estimation of body exposure to drug after administration, which is reflected by the area under the plasma concentration-time curve (AUC). The AUC was first computed with the trapezoidal method. Thereafter, the area under the first moment curve (AUMC), mean residence time (MRT) and mean absorption time (MAT) were calculated as follows:

Using these estimates, the SC bioavailability, F, the plasma clearance (CL) were estimated as follows:

Similarly, the terminal half-life, T_{1/2, Terminal}, was best-fit to the log-linear decay observed in each individual over the last three time points.

Regarding the compartmental model-based analysis, the volume of distribution of the plasma compartment (V_d), the elimination rate constant (k_elimination), the transfer rate constant from plasma to tissue (k_{plasma➜tissue}) and the transfer rate constant from tissue back to plasma (k_{tissue➜plasma}) were first solved from the two-compartmental IV model. This IV model was then used to construct the four-compartmental SC analysis under the assumption that V_d and k_elimination remain constant from IV to SC. This enabled fitting of additional constants in the SC model including an apparent elimination rate constant from interstitial fluid (ISF) (k_{ISF_elimination}), which was used to account for the observed bioavailability, F. If either k_{ISF_elimination} or k_{SC_site->ISF} were rate limiting (ie. much greater than the other), than the model would reduce to a one-phase absorption; however, a one-phase absorption model was unable to fit the late peak times observed. This suggested that k_{ISF_elimination} and k_{SC_site->ISF} are on the same order of magnitude; therefore, the assumption was made that k_{ISF_elimination} = k_{SC_site->ISF}, which enabled good fitting to each mouse. From these best-fit curves generated in SAAM II, the peak concentration, C_max, and peak time, t_max, were extracted from calculated points of each fit after increasing the Minimum Number of Calculation Intervals to 500. For comparison with the noncompartmental CL, the clearance from the compartmental model was solved by the following equation: