Biological assessment of triazine dendrimer: toxicological profiles, solution behavior, biodistribution, drug release and efficacy in a PEGylated, paclitaxel construct

Abstract

The physicochemical characteristics, in vitro properties, and in vivo toxicity and efficacy of a third generation triazine dendrimer bearing approximately nine 2 kDa polyethylene glycol chains and twelve ester linked paclitaxel groups are reported. The hydrodynamic diameter of the neutral construct varies slightly with aqueous solvent ranging from 15.6 to 19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former approximately 40 kDa mass consistent with expectation, and the latter 400 kDa mass consistent with a decameric structure and the observed hydrodynamic radii. HPLC can be used to assess purity as well as paclitaxel release, which is insignificant in organic solvents or aqueous solutions at neutral and low pH. Paclitaxel release occurs in vitro in human, rat, and mouse plasma and is nonlinear, ranging from 7 to 20% cumulative release over a 48 h incubation period. The construct is 2-3 orders of magnitude less toxic than Taxol by weight in human hepatocarcinoma (Hep G2), porcine renal proximal tubule (LLC-PK1), and human colon carcinoma (LS174T) cells, but shows similar cytotoxicity to Abraxane in LS174T cells. Both Taxol and the construct appear to induce caspase 3-dependent apoptosis. The construct shows a low level of endotoxin, is not hemolytic and does not induce platelet aggregation in vitro, but does appear to reduce collagen-induced platelet aggregation in vitro. Furthermore, the dendrimer formulation slightly activates the complement system in vitro due most likely to the presence of trace amounts (<1%) of free paclitaxel. An animal study provided insight into the maximum tolerated dose (MTD) wherein 10, 25, 50, and 100 mg of paclitaxel/kg of construct or Abraxane were administered once per week for three consecutive weeks to non tumor bearing athymic nude mice. The construct showed in vivo toxicity comparable to that of Abraxane. Both formulations were found to be nontoxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-h-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort.

Figure 1

Gas phase simulations of 1 with nine PEG chains reveal opportunities for hydrophobic patch formation on one face derived largely from paclitaxel groups. Each PEG chain is colored violet with purple used to indicate the site of attachment to the dendrimer. Dendrimer and paclitaxel are indicated using conventional colors (C=grey, H=white, N=blue, O=red). Little of the triazine dendrimer is apparent.

Figure 2

Chromatogram of 1 showing free paclitaxel (~9 min) and a series of peaks (attributed to differing degrees of PEGylation) corresponding to dendrimer 1.

Figure 3

Cumulative release (in %) of paclitaxel from 1 in human, rat, and mouse plasma.

Figure 4

Biodistribution and tumor localization of 1 in SCID mice bearing PC-3 xenografts showing tumor/muscle ratios at different time points, respectively.

Figure 5

Therapeutic efficacy of 1 in SCID mice bearing PC-3-h-luc xenografts. (A): Tumor volume changes measured by caliper during the 10-week treatment period (tumor volume ratio = tumor size at a given time/tumor size at day 4 post treatment). (B) Statistical comparison between the treatment groups (pooled data from 100s, 100d, 200s, and 200d shown as black columns) and the PBS control. Significant therapeutic efficacy (p < 0.0001) was seen starting from day 50 post treatment. (C) Expanded portion of the treatment group data in (A). The abbreviations reflect the amount of paclitaxel in mg PTX/kg (100 or 200) and whether a single (s) or double (d) administration was used.

Figure 6

BLI evaluation of the therapeutic efficacy of 1 in SCID mice bearing PC-3-h-luc xenografts. (A): Representative BLI images of four treatment groups and PBS control. The photon intensity of the BLI images was shown on the same scale. (B) Comparative presentation of BLI signals of the four treatment groups. The relative BLI signals at the given week were normalized to the data at week 3 (set at 100%) for all individual mice. (C) Absolute BLI photon intensity quantification of the five tumor-bearing mice shown in (A).

Figure 7

Correlations between the tumor size (caliper measures) and tumor cell viability (BLI photon intensity).

Chart 1

Dendrimer 1.