High-performance liquid chromatography (HPLC)-grade acetonitrile was obtained from Burdick & Jackson (Muskegon, MI, USA), reagent-grade formic acid was obtained from EM Science (Gibbstown, NJ, USA), and paclitaxel bulk drug was purchased from Sigma-Aldrich (St. Louis, MO, USA). Commercially available paclitaxel (Cremophor EL:ethanol) was manufactured by Bristol-Myers Squibb (New York, NY, USA). Other chemicals were either made in-house (Genentech, Inc., South San Francisco, CA, USA) or purchased from Sigma-Aldrich. The water purification system used was a Millipore Milli-Q system (Billerica, MA, USA).
Powder X-ray diffraction pattern and particle size determination
Powder X-ray diffraction (PXRD) patterns were recorded at room temperature with a Rigaku (The Woodlands, TX, USA) MiniFlex II desktop X-ray powder diffractometer. Radiation of Cu Kα at 30 kV and −15 mA was used with 2θ increment rate of 3°/min. The scans were run over a range of 2° to 40° 2θ with a step size of 0.02° and a step time of 2 s. Powder samples were placed on a flat silicon zero background sample holder.
The particle size distribution of the nanosuspension was measured by using a Nanotrac (Montgomeryville, PA, USA) instrument. Triplicates were measured for each sample, and the average was used for the final particle size distribution. The particle size distribution was calculated based on the general purpose (normal sensitivity) analysis model and the following refractive indices (RIs): particle RI, 1.58; absorption, 1.0; and dispersant RI, 1.38.
Formulation preparation for paclitaxel IV crystalline nanosuspension and stability evaluation
A bench scale wet milling method was developed for particle size reduction and has been previously described . Briefly, a paclitaxel stock nanosuspension formulation (20 mg/mL) was prepared by mixing paclitaxel with an appropriate amount of glass beads and vehicle containing 0.1% (w/w) Cremophor EL in phosphate saline (pH 7.4) in a scintillation vial. The mixture was stirred at 1,200 rpm for a period of 24 h with occasional shaking. The resulting stock formulation was diluted to the target concentration with vehicle and then harvested. Paclitaxel concentrations were verified by a HPLC assay. Analysis of milled paclitaxel particles was performed using a Nanotrac (Montgomeryville, PA, USA) instrument. An assessment of form change in pre- and post-milling samples was performed using PXRD.
The rate of dissolution of paclitaxel in nanosuspension is expected to be higher compared to regular suspension due to the reduction of particle size. The Noyes and Whitney equation (Equation 1
) was used in order to assess the impact of particle size reduction on dissolution rate and is described as follows:
where dC/dt is the dissolution rate, D is the solute diffusion coefficient, V is the volume of the dissolution medium, h
is the diffusion boundary thickness, S is the surface area of the solute, C
is the saturation solubility of the solute, and C
(t) is the bulk solute concentration. The stability of the crystalline nanosuspension was monitored for a period of 3 weeks for particle size, PXRD, and chemical stability (by HPLC). Paclitaxel plasma solubility was determined by adding excess amount of paclitaxel bulk drug into 0.5 mL of rodent plasma (obtained in-house) which was allowed to equilibrate at 37°C on a rotary shaker for a period of 24 h. Excess drug was then removed by centrifugation which was followed by protein precipitation, and the concentration was measured by HPLC with an external standard.
Efficacy and pharmacokinetic study in xenograft mice
Briefly, 2.5 million Calu-3 non-small cell lung cancer cells were resuspended in Hank's balanced salt solution and implanted intradermally into the hind flank of female SCID-bg mice (Charles River Laboratories, Hollister, CA, USA). When tumor volumes reached approximately 150 to 300 mm3, mice were randomly assigned to three treatment groups. Treatment groups were administered one intravenous dose every 4 days of either vehicle (Cremophor EL:ethanol 1:1, saline; n = 10), paclitaxel formulated in Cremophor vehicle (n = 15), or paclitaxel formulated in nanosuspension (n = 15). A total of three doses were given during the course of the study. The paclitaxel dose was selected in an attempt to match as best possible clinically relevant exposures and at the same time provide robust anti-tumor efficacy when delivered with the commercial formulation (Cremophor EL:ethanol 1:1). Tumor volumes were measured in two dimensions (length and width) using Ultra Cal-IV calipers (Model 54-10-111, Fred V. Fowler Company, Inc., Newton, MA, USA). The following formula was used with Excel v11.2 (Microsoft Corporation, Redmond, WA, USA) to calculate tumor volume (TV): TV (mm3) = (length × width2) × 0.5. Tumor sizes and body weights were recorded twice weekly, and the mice were regularly observed over the course of the study. Mice were euthanized if their tumor volume exceeded 2,000 mm3 or if their body weight dropped by more than 20% of the starting weight. At end of the study, mice in both paclitaxel groups were given a final dose of paclitaxel, and blood (collected by terminal cardiac puncture and plasma-harvested) and tissues (liver, spleen, and tumor) were collected at various time points (10 min, 30 min, 2 h, 4 h, and 8 h post-dose). Three mice were taken down at each time point, and biological samples were frozen at −70°C until sampling. Paclitaxel concentrations in plasma and tissues were measured by a liquid chromatography tandem mass spectrometry (LC/MS/MS) assay. The study was conducted in accordance with the institutional guidelines for humane treatment of animals and was approved by the IACUC of Genentech.
LC/MS/MS assay for the determination of paclitaxel
Concentrations of paclitaxel in mouse plasma, tumor, liver, and spleen were determined by a LC/MS/MS assay. Tumor, liver, and spleen tissue samples were diluted 4-fold with water and homogenized by using a FastPrep-24 bead beater (MP Biomedicals, Solon, OH, USA). The plasma and tissue homogenate samples were prepared for analysis by placing a 25-μL aliquot into a 96-well plate followed by the addition of 5 μL of internal standard (docetaxel, 2 μg/mL in 50:50, v/v, DMSO:water) and 200 μL acetonitrile. The samples were vortexed and centrifuged at 1,600 g for 15 min at room temperature, 50 μL of the supernatant was diluted with 150 μL of water, and 5 μL of the solution was injected onto a Kinetex XB C-18 (30 × 2.1 mm, 2.6 μm) analytical column (Phenomenex, Torrance, CA, USA).
An Agilent 1290 Infinity HPLC system (Agilent, Santa Clara, CA, USA) was equipped with a controller, two pumps, a column compartment, and a degasser. The column was maintained at 40°C by the column compartment. This system was coupled to an API 5500 Qtrap mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with a turbo-electrospray interface in positive ionization mode. The aqueous mobile phase was water with 0.1% formic acid (A), and the organic mobile phase was acetonitrile with 0.1% formic acid (B). The gradient was as follows: starting at 15% B and increased to 95% B for 0.6 min, maintained at 95% B for 0.1 min, then decreased to 15% B within 0.1 min. The total flow rate was 1.4 mL/min. Data was collected using multiple reaction monitoring (MRM) with transitions m/z 854.4 → 104.9 for paclitaxel and m/z 808.5 → 527.2 for docetaxel (internal standard). The calibration curve, which ranged from 0.03 to 24 μM for paclitaxel, was fitted to a 1/x weighted quadratic regression model. This calibration curve was used to quantitate paclitaxel concentration levels in the plasma, tumor, liver, and spleen samples.
Pharmacokinetic parameters were estimated by non-compartmental methods as described by Gibaldi and Perrier  using WinNonlin version 3.2 (Pharsight Corporation, Mountain View, CA, USA). Tissue to plasma ratios were determined by dividing the AUC0-8 (area under the concentration-time profile from 0 to 8 h) of the tissue of interest by the AUC0-8 of plasma.
The percent tumor growth inhibition (%TGI) was calculated on the last day of the study (day 17) using the following formula as previously described [36
TVvehicle is the tumor volume for the vehicle-treated animals on day 17, TVinitial is the initial tumor volume at the start of the treatment, and TVtreatment is the tumor volume of the treatment groups on day 17. Normalized efficacy was determined with respect to plasma and tumor exposures for both Cremophor EL:ethanol and nanosuspension delivery. Normalized efficacy was determined by dividing TGI by either plasma or tumor AUC0-8.