Design, synthesis and biological assessment of a triazine dendrimer with approximately 16 Paclitaxel groups and 8 PEG groups

Abstract

The synthesis and characterization of a generation three triazine dendrimer that displays a phenolic group at the core for labeling, up to eight 5 kDa PEG chains for solubility, and 16 paclitaxel groups is described. Three different diamine linkers--dipiperidine trismethylene, piperazine, and aminomethylpiperidine--were used within the dendrimer. To generate the desired stoichiometric ratio of 8 PEG chains to 16 paclitaxel groups, a monochlorotriazine was prepared with two paclitaxel groups attached through their 2'-hydroxyls using a linker containing a labile disulfide. This monochlorotriazine was linked to a dichlorotriazine with aminomethylpiperidine. The resulting dichlorotriazine bearing two paclitaxel groups could be reacted with the eight amines of the dendrimer. NMR and MALDI-TOF confirm successful reaction. The eight monochlorotriazines of the resulting material are used as the site for PEGylation affording the desired 2:1 stoichiometry. The target and intermediates were amenable to characterization by (1)H and (13)C NMR, and mass spectrometry. Analysis revealed that 16 paclitaxel groups were installed along with 5-8 PEG chains. The final construct is 63% PEG, 22% paclitaxel, and 15% triazine dendrimer. Consistent with previous efforts and computational models, 5 kDa PEG groups were essential for making the target water-soluble. Molecular dynamics simulations showed a high degree of hydration of the core, and a radius of gyration of 2.8 ± 0.2 nm. The hydrodynamic radius of the target was found to be 15.8 nm by dynamic light scattering, an observation indicative of aggregation. Drug release studies performed in plasma showed slow and identical release in mouse and rat plasma (8%, respectively). SPECT/CT imaging was used to follow biodistribution and tumor uptake. Using a two component model, the elimination and distribution half-lives were 2.65 h and 38.2 h, respectively. Compared with previous constructs, this dendrimer persists in the vasculature longer (17.33 ± 0.88% ID/g at 48 h postinjection), and showed higher tumor uptake. Low levels of dendrimer were observed in lung, liver, and spleen (~6% ID/g). Tumor saturation studies of small prostate cancer tumors (PC3) suggest that saturation occurs at a dose between 23.2 mg/kg and 70.9 mg/kg.

Chart 1

Paclitaxel-conjugated triazine dendrimers 1-3. Both 1 and 3 have disulfide linkers. Colors reflect those used in Scheme 1. PTX=paclitaxel.

Figure 1

MD simulations show differences in size and solvent accessible surface area (SASA) for 4 pegylated with 2kDa PEG and 5kDa PEG (1). Radial distribution functions show that 2kDa PEG is insufficient in masking the hydrophobic dendrimer.

Figure 2

The light-scattering distribution analysis of prodrug 1

Chart 1

Additional HPLC standards with sites of cleavage of 1 indicated with arrows.

Figure 3

(A) Tissue distribution of prodrug 1 in normal BALB/c mice. Data are presented as %ID/g ± s.d. (n = 4). (B) Pharmacokinetic parameters of prodrug 1 in normal BALB/c mice. (C) Excretion profile of prodrug 1 from BALB/c mice (n = 4).

Figure 4

Tumor Saturation Dose Evaluation of ¹²⁵I-1 in PC-3 Tumor-bearing Mice: 1.7 mg/kg (blue), 23.2 mg/kg (red), 70.9 mg/kg (purple), and 111.3 mg/kg (green). (A) Representative transaxial SPECT/CT images of PC3 tumor in SCID mice (48 h p.i.). Tumors are indicated by white arrows. (B) Tumor uptake of ¹²⁵I-1 versus tumor size. Tumors smaller than 100 mm³ were selected for the evaluation. (Number of tumors evaluated in each group: 7 (1.7 mg/kg); 5 (23.2 mg/kg); 8 (70.9 mg/kg); 5 (111.3 mg/kg). (C) Tumor uptake levels of the 4 dosing groups in tumors smaller than 40 mm³. (Number of tumors evaluated in each group: 4 (1.7 mg/kg); 5 (23.2 mg/kg); 8 (70.9 mg/kg); 4 (111.3 mg/kg)). SUV is standardized uptake value of the labeled prodrug.

Scheme 1. Retrosynthetic analysis

Target 1 derives from a two-step PEGylation procedure of 4. Intermediate 4 results from reaction of the functionalized dichlorotriazine bearing two paclitaxel groups (PTX), 5, with dendrimer 6. Dendrimer 6 comes from click reaction of 7 which derives from some of the building blocks identified.

Scheme 2

Synthesis of the generation two dendrimer

Scheme 3

Synthesis of the dichlorotriazine.