MAC-321, a novel taxane with greater efficacy than paclitaxel and docetaxel in vitro and in vivo

In vitro tubulin polymerization assay. Tubulin polymerization assays were conducted at 24°C using 1.5 mg/ml of purified bovine brain MAP-free tubulin in the presence of vehicle control (▴) or the indicated concentrations of DTX (closed symbols) or MAC-321 (open symbols) as shown. Turbidity was measured by absorbance (340 nm) for up to 60 min.

Immunofluorescence staining of α-tubulin in KB-3-1 cells treated with PTX and MAC-321. Cells were treated for 20 h with vehicle (A), 8 nm vinblastine (B), 1.25 nm MAC-321 (C), 12.5 nm MAC-321 (E), 4 nm PTX (D), or 40 nm PTX (F). After fixation, cells were incubated with an anti-α-tubulin antibody followed by a FITC-conjugated secondary antibody and stained with propidium iodide to visualize DNA. Images were overlaid electronically after cells were examined by fluorescent microscopy for tubulin and propidium iodide staining. Insets in panels A, B, C, and E were electronically enlarged to demonstrate tubulin morphology. Arrowheads in panels B, C, E, and F indicate altered microtubule morphology.

Expression of MDR1 mRNA and protein in a panel of tumor cell lines. RNA (A) or membrane proteins (B) was prepared from non-selected breast, lung, colon, or ovarian cell lines or from parental (KB-3-1) cell lines selected for resistance to colchicine (KB-8-5) or vinblastine (KB-V1). A, levels of MDR1 mRNA were assessed by real-time RT-PCR. Columns, mean relative expression from two experiments of MDR1 mRNA normalized to β-actin mRNA as described in “Materials and Methods”; bars, SD. Expression is represented on a logarithmic scale with the lowest expressing cell line, NCI-H838, set at 1. B, P-glycoprotein was detected by immunoblotting methods using an anti-MDR1 antibody.

Accumulation of MAC-321 and PTX in KB-3-1 cells. The intracellular accumulation of MAC-321 in KB-3-1, KB-8-5, and KB-V1 cells was performed as described in “Materials and Methods.” Cells were incubated with [¹⁴C]-MAC-321 (104 mCi/mmol) and [¹⁴C]-PTX (74 mCi/mmol) at a final concentration of 500 nm (0.051 and 0.039 μCi/ml, respectively) for 2 h at 37°C. Radioactivity in the cell lysates was determined by liquid scintillation counting and was normalized for protein content. Data are reported as mean pmol drug/mg of total protein from triplicate wells.

Efficacy of MAC-321 in PTX-sensitive xenograft tumor models in vivo by i.v. and p.o. administration. Female nu/nu mice were given injections of 1.5 × 10⁶ Lox melanoma cells, 5 × 10⁶ KB-3-1 cells, or 8 × 10⁶ A375SM melanoma cells. Animals bearing Lox melanoma tumors approximately 100 mg in size were treated i.v. with vehicle or 10–70 mg/kg MAC-321 (A) or 10–200 mg/kg p.o. (B) on day 1. Tumor growth in all cases was determined approximately every 7 days. C and D, animals bearing KB-3-1 or A375SM staged tumors were treated with vehicle, 70 mg/kg MAC-321 p.o., or 70 mg/kg MAC-321 i.v. on day 1. In KB-3-1 xenografts, PTX was given i.v. at 60 mg/kg on day 1 or 20 mg/kg on days 1, 5, 9, and 13 (C). In A375SM xenografts, PTX was dosed i.v. at 60 mg/kg on days 1, 5, and 9 only (D). Statistical significance was determined by Student's two-tailed t test for all doses and test agents compared with the vehicle control group. *, P ≤ 0.01

Activity of MAC-321 (i.v. and p.o.) in PTX-resistant xenograft tumor models in vivo. Female nu/nu mice were given injections of 6 × 10⁶ DLD-1 colon carcinoma cells, 5 × 10⁶ KB-8-5 cells, or five fragments of MX-1W breast carcinoma. Animals bearing DLD-1 staged tumors were treated i.v. (A) or p.o. (B) with vehicle or at the doses of MAC-321 indicated on the day after reaching approximately 100 mg in size. In addition, DLD-1 xenografts were also treated i.v. with 25 mg/kg of DTX on days 1, 5, and 9. Animals bearing KB-8-5 (C) or MX-1W (D) staged tumors were treated with either vehicle, 70 mg/kg MAC-321 p.o., or 70 mg/kg MAC-321 i.v. on day 1. Sixty milligrams per kilogram PTX were administered i.v. on days 1, 5, and 9 (C and D). *, P ≤ 0.01 and #, P ≤ 0.02 as determined by Student's two-tailed t test.