**2.6 Oral administration of paclitaxel formulations**

Paclitaxel formulations in Tables 2 and 3 were liquefied at 37 ~ 50 C and administered orally at doses of 25, 50, 75, and 100 mg/kg with a blunt needle via the esophagus into the stomach. The male ICR mice were fasted for 8 h prior to oral administration except for the group G3. Taxol® prepared by dissolving paclitaxel in an equivolume mixture of Cremophor EL and ethanol at 6 mg/ml (Taxol®) was diluted by 6 times with the saline solution, and was administered via bolus tail-vein injection at a dose of 10 mg/kg as a positive control. Blood samples were collected at various time points (n=6) after drug administration, and were stored at -70 C until analysis.

#### **2.7 Analysis of paclitaxel concentrations in blood**

Whole blood (200 μl) was spiked with irbesartan (0.5 μg/ml; internal standard), mixed and added to acetonitrile (400 μl) to precipitate proteins. After centrifugation at 14,000 RCF for 20 min, the supernatant was collected and mixed with the mobile phase to adjust the volume to 0.6 ml. Ten microliters of the blood was injected into the LC/MS/MS system. Analyses were performed with a Thermo-Finnigan Discovery Max LC/MS/MS (San Jose, CA, USA). The LC system was performed at 35 C on a Capcellpak C18 column (150 X 2.0 mm i.d., 5 μm particle size, Shisheido, Japan) equipped with a Zorbax SB-Aq (12.5 X 2.1 mm i.d.) guard column. The mobile phase consisted of 55 % acetonitrile, 0.08 % formic acid and 44.92 % water, and the flow rate was 0.4 ml/min. The instrument was operated in SRM mode (positive ion), monitoring the ion transitions from m/z 854 285 (paclitaxel) and m/z 429 195 (internal standard). The paclitaxel LC/MS/MS assay was linear over the range of 2 ~ 1000 ng/ml with a lower quantitation limit of 2 ng/ml in blood. Paclitaxel

Development, Optimization and Absorption Mechanism of

anhydrous crystalline forms, respectively.

ingredients were added to prepare the formulations in Table 3.

**3.2 Physical properties of oral paclitaxel formulations** 

DHP107, Oral Paclitaxel Formulation for Single-Agent Anticancer Therapy 363

Fig. 2. Differential Scanning calorimetry, x-ray diffraction and scanning electron microscopy

we confirmed that Paclitaxel from Samyang Genex and Indena were in amorphous and

Due to the differences in the grain size and crystalline forms, we used two different procedures in preparing the oral formulations. Genexol 131 was readily soluble in all of the lipid mixtures in Table 2 when sonicated for *ca*. 30 s. Genexol 183, which had larger grains than Genexol 131, and paclitaxel from Indena did not dissolve in the lipid mixture. In these cases, paclitaxel was solubilized completely in the mixture of methylene chloride and triglyceride. Methylene chloride was evaporated completely from the mixture before other

Oral paclitaxel formulations existed as oil/wax mixtures at ambient temperatures, but formed clear liquid at 37 C except for the Formulation T12 (Table 2). The T12 formulation existed as solid wax at ambient temperatures. Thermal behavior of the formulations

The thermograms were identical with or without paclitaxel in the formulations, and those with paclitaxel are shown in the Figure. Tween 80, liquid at room temperature, did not show any phase transition in the temperature range from 0 to 60 C. Lamellar crystalline-to-fluid isotropic, or the chain melting, phase transition of monoolein was observed at 32 C, which is similar to the values reported for pure monoolein (Briggs et al., 1996; Qiu & Caffrey, 2000) or Myverol 18-99K (Clogston, 2000) in the literature. The chain melting transition of monoolein for the monoolein/Tween 80 (55.0:16.5 by weight) mixture was observed at 26 C, since Tween 80 lowered the transition temperature of monoolein. In the formulations T2, T4 and T6, only the chain melting transition of monoolein was observed because triacetin,

containing different triglycerides was investigated by performing DSC (Figure 3A).

of paclitaxel obtained from of Genexol (upper panels) and Indena (lower panels).

concentrations in blood were calculated based on a standard curve of paclitaxel in blank pooled animal blood with the internal standard.

#### **2.8 Tumor experiment**

Suspension of NCI-H358 human non-small cell lung cancer cells (1.2×107 cells/mouse), purchased from American Type Culture Collection (ATCC, Manassas, VA), was injected subcutaneously to the dorsal flank of male Balb/c athymic mice (8 weeks). When the tumor volume (length × width × height × 0.5236) reached *ca*. 100 mm3 in 10 days after the injection (day 0), the experimental groups were divided at random into three groups (n=8). On days 1, 2, 3, 4 and 5, G2 formulation (DHP107) was administered orally at a dose of 50 mg/kg (G2). For controls, mice injected intravenously with diluted Taxol® (Taxol, 10 mg/kg) and fed orally with the vehicle only (eG2) on days 1, 2, 3, 4 and 5 were also observed.
