Combined delivery of paclitaxel and tanespimycin via micellar nanocarriers: pharmacokinetics, efficacy and metabolomic analysis

Abstract

Background: Despite the promising anticancer efficacy observed in preclinical studies, paclitaxel and tanespimycin (17-AAG) combination therapy has yielded meager responses in a phase I clinical trial. One serious problem associated with paclitaxel/17-AAG combination therapy is the employment of large quantities of toxic organic surfactants and solvents for drug solubilization. The goal of this study was to evaluate a micellar formulation for the concurrent delivery of paclitaxel and 17-AAG in vivo.

Methodology/principal findings: Paclitaxel/17-AAG-loaded micelles were assessed in mice bearing human ovarian tumor xenografts. Compared with the free drugs at equivalent doses, intravenous administration of paclitaxel/17-AAG-loaded micelles led to 3.5- and 1.7-fold increase in the tumor concentrations of paclitaxel and 17-AAG, respectively, without significant altering drug levels in normal organs. The enhanced tumor accumulation of the micellar drugs was further confirmed by the whole-body near infrared imaging using indocyanine green-labeled micelles. Subsequently, the anticancer efficacy of paclitaxel/17-AAG-loaded micelles was examined in comparison with the free drugs (weekly 20 mg/kg paclitaxel, twice-weekly 37.5 mg/kg 17-AAG). We found that paclitaxel/17-AAG-loaded micelles caused near-complete arrest of tumor growth, whereas the free drug-treated tumors experienced rapid growth shortly after the 3-week treatment period ended. Furthermore, comparative metabolomic profiling by proton nuclear magnetic resonance revealed significant decrease in glucose, lactate and alanine with simultaneous increase in glutamine, glutamate, aspartate, choline, creatine and acetate levels in the tumors of mice treated with paclitaxel/17-AAG-loaded micelles.

Conclusions/significance: We have demonstrated in the current wok a safe and efficacious nano-sized formulation for the combined delivery of paclitaxel and 17-AAG, and uncovered unique metabolomic signatures in the tumor that correlate with the favorable therapeutic response to paclitaxel/17-AAG combination therapy.

Figure 1. Paclitaxel/17-AAG-loaded PEG-DSPE/TPGS mixed micelles.

A, the structural scheme of the dual drug-loaded micelles. B, the hydrodynamic diameter of the dual drug-loaded micelles. Number (%) shows among the total number of the counted particles in a sample, the percentage of the number of the particles within each size class. The result shows representative data obtained from 3 independent measurements.

Figure 2. Simultaneous quantification of paclitaxel and 17-AAG by HPLC chromatography.

Representative chromatograms of pure paclitaxel and 17-AAG (green trace), an extract of drug-spiked liver tissue (blue trace) and a blank liver extract (black trace) are shown at 221 nm (A), 333 nm (B) and 281 nm (C), while the peaks of interest at each detection wavelength are shown in red. The retention time of paclitaxel, 17-AAG and α-naphthoflavone was 27.6 min, 36.8 min and 39.3 min, respectively. The concentration of paclitaxel and 17-AAG was both 10 µM, and α-naphthoflavone was used at 20 µM as an internal standard.

Figure 3. Pharmacokinetics of paclitaxel/17-AAG-loaded micelles in nude mice bearing human ovarian tumor SKOV-3 xenografts.

The dual drug-loaded micelles were i.v. administered at the doses of 20 mg/kg paclitaxel and 37.5 mg/kg 17-AAG. For the free drug-treated group, the mice received the same combined doses of free paclitaxel and 17-AAG dissolved in DMSO. Each data point was the average+SE, n = 4 mice per group. A, the micellar formulation resulted in over 10-fold increase in paclitaxel concentrations in plasma. The plasma concentration of paclitaxel following the free drug administration was below the detection limit at 4 h. B, the micellar formulation resulted in over 3-fold increase in 17-AAG concentrations in plasma. The plasma concentration of 17-AAG was below the detection limit at 4 h for both groups. C, the micellar formulation caused a 3.5-fold increase of paclitaxel (***, p = 0.0001) in the tumor without significant affecting the drug distribution to normal organs. D, the micellar formulation caused a 1.7-fold increase of 17-AAG (***, p = 0.0005) in the tumor without significant affecting the drug distribution to normal organs.

Figure 4. The NIR imaging of ICG-labeled micelles in nude mice bearing SKOV-3 xenografts.

A, the stability of ICG at 37°C was greatly enhanced when it was loaded within PEG-DSPE/TPGS mixed micelles. In contrast, free ICG was highly unstable in aqueous solution. Results show representative data obtained from three independent experiments and are reported as the average+SD (n = 3). B, ICG-labeled micelles were i.v. injected at an ICG dose of 2 mg/kg. The control mice received an equal dose of ICG, which was freshly prepared in aqueous solution. Immediately following the injection (time 0) and at 1, 2, 4, 6, 24 and 48 h, the spectral fluorescence signals of the whole body images (800 nm channel) were obtained using the LI-COR Odyssey imaging system. Representative images are shown, n = 3 mice per group.

Figure 5. Paclitaxel/17-AAG-loaded micelles potentiate the anticancer efficacy of the drugs in nude mice bearing SKOV-3 xenografts.

Mice were randomized, and treatment was initiated on day 0. The mice were i.v. dosed with the combined doses of paclitaxel (20 mg/kg) and 17-AAG (37.5 mg/kg) either as the free drugs dissolved in DMSO, or the dual drug-loaded micelles on days 0, 7 and 14. On days 3, 10 and 17, the mice in these two groups also received 17-AAG (37.5 mg/kg) as the free drug or the drug-loaded micelles. The untreated mice served as controls. A, paclitaxel and 17-AAG in their free forms significantly delayed the tumor growth but was less effective than the micellar formulation, which caused near-complete arrest of tumor growth. Starting day 22, the average tumor sizes among all three groups were significantly different (p<0.05). Each data point was the average+SE, n = 6 mice per group. B, the tumor weights on day 43 from all three groups showed drastic differences. (*, p<0.05; ***, p<0.0005) C, phosphorylation of Akt and its immediate downstream substrate GSK3α was markedly inhibited in the tumors of the micellar drug-treated mice, but remained highly activated in the tumors of the free drug-treated mice. The total levels of Akt and GSK3α were unaffected by either treatment. β-Actin was used as a loading control. The results show representative data obtained from 3 independent analyses. D, immunohistochemical staining of Ki-67 indicated a large number of proliferating tumor cells in the untreated and free drug-treated mice, but few in the micellar drug-treated mice (original magnification ×200). E, the average body weight of mice remained constant in all groups throughout the study.

Figure 6. Metabolomic analysis of the mouse tumor extracts.

A, scores plot of the OPLS-DA model for the untreated and treated tumors, which shows the group separation among the untreated (square), free drug-treated (circle) and micellar drug-treated (diamond) tumors. The percentages of explained variation modeled for the first two components (OPLS1 and OPLS2) are displayed on the axes. B, there was no clear correlation between the OPLS1 scores and their corresponding tumor weights. C, Student’s t-test filtered difference NMR spectra (p<0.05) of tumors treated with paclitaxel/17-AAG combination in the free forms (top) and in the micellar formulation (bottom). The positive peaks correspond to the metabolites increased in concentration relative to the untreated tumors, whereas the negative peaks are from the metabolites decreased. The identified metabolites are: Glc, glucose; Cho, choline; Crt, creatine; Asp, aspartate; Gln, glutamine; Glu, glutamate; Ala, alanine; Lac, lactate.