Doxil — The First FDA-Approved Nano-Drug: From an Idea to a Product - Harnessing Biomaterials in Nanomedicine: Preparation, Toxicity, and Applications - page 354

12.3.3 OLV-DOX Pharmacokinetics, Biodistribution, and

in vivo Imaging in Humans

The PK and BD study in humans (Gabizon et al., 1991) included 19
patients receiving OLV-DOX. It was carried out within the framework
of a Phase I clinical trial (Gabizon et al., 1989a, 1989b; Barenholz et
al., 1990). The formulation of DOX-OLV tested consisted of the DOX-
OLV described above. Plasma clearance of total drug extracted from
the plasma after DOX-OLV infusion followed a bi-exponential decay
curve with a pattern and PK parameters similar to that reported
(and confirmed by us) for free DOX. The plasma concentration of
drug circulating in liposome-associated form was also determined
using the method described by Druckmann et al. (1989), which
is based on separation of free and liposome-associated drug by
the cation exchanger Dowex-50 (which selectively binds the free
drug). These special studies were performed in seven patients.
Liposome-associated drug was found to be rapidly cleared from
plasma; its ratio to nonliposome-associated drug appeared to
correlate with liver reserve, with highest ratios in patients with
normal liver function. Liposomes' clearance was measured after
free drug and protein-associated drug were removed by Dowex-
50 cation-exchange resin (Druckmann et al., 1989). The remaining
plasma that contained the OLV-DOX was thereafter processed for
HPLC drug analysis as detailed elsewhere (Gabizon et al., 1991a).
Measurements of total and liposome-associated plasma doxorubicin
were obtained. The levels of free and protein-bound drugs were
inferred by subtracting the concentration of liposome-associated
drug from that of total doxorubicin. In three of these patients, we
also measured the concentration of PG in plasma to follow the
clearance of liposomes (Gabizon et al., 1991a; Amselem et al.,
1993a). PG was selected as a liposome marker because of its very
low concentration in human plasma (less than 5 nmoles/mL) relative
to the concentration of total phospholipids (2,000-4,000 nmoles/
mL). Phosphatidylethanolamine (PE) was chosen as an internal
standard due to its absence in the DOX-OLV formulation and its
being in a concentration range of similar order of magnitude to the
phospholipids infused as OLV-DOX. Plasma samples were extracted
after a complete trinitrophenylation of the plasma aminolipids (PE
and phosphatidylserine) and of DOX by trinitrobenzene sulfonate
(TNBS). The step of trinitrophenylation was essential to optimize