AMXI-5001, a novel dual parp1/2 and microtubule polymerization inhibitor for the treatment of human cancers

Abstract

Poly (ADP-ribose) polymerase (PARP) has recently emerged as a central mediator in cancer resistance against numerous anticancer agents to include chemotherapeutic agents such as microtubule targeting agents and DNA damaging agents. Here, we describe AMXI-5001, a novel, highly potent dual PARP1/2 and microtubule polymerization inhibitor with favorable metabolic stability, oral bioavailability, and pharmacokinetic properties. The potency and selectivity of AMXI-5001 were determined by biochemical assays. Anticancer activity either as a single-agent or in combination with other antitumor agents was evaluated in vitro. In vivo antitumor activity as a single-agent was assessed in a triple-negative breast cancer (TNBC) model. AMXI-5001 demonstrates comparable IC50 inhibition against PARP and microtubule polymerization as clinical PARP inhibitors (Olaparib, Rucaparib, Niraparib, and Talazoparib) and the potent polymerization inhibitor (Vinblastine), respectively. In vitro, AMXI-5001 exhibited selective antitumor cytotoxicity across a wide variety of human cancer cells with much lower IC50s than existing clinical PARP1/2 inhibitors. AMXI-5001 is highly active in both BRCA mutated and wild type cancers. AMXI-5001 is orally bioavailable. AMXI-5001 elicited a remarkable In vivo preclinical anti-tumor activity in a BRCA mutated TNBC model. Oral administration of AMXI-5001 induced complete regression of established tumors, including exceedingly large tumors. AMXI-5001 resulted in superior anti-tumor effects compared to either single agent (PARP or microtubule) inhibitor or combination with both agents. AMXI-5001 will enter clinical trial testing soon and represents a promising, novel first in class dual PARP1/2 and microtubule polymerization inhibitor that delivers continuous and synchronous one-two punch cancer therapy with one molecule.

Keywords: AMXI-5001; BRCA; PARP inhibitor; breast cancer; cancer therapy; homologous recombination; malignancy; microtubule inhibitor; synthetic lethality.

Figure 1

AMXI-5001 is a potent PARP1/2 inhibitor. (A) Structure of AMXI-5001 HCl. (B-D) The inhibitory action of the test compounds towards PARP1 (B, C) and PARP2 (D) was determined using a commercially available microplate assay (Universal Colorimetric PARP Assay from Trevigen, Inc) for PARP1 and (BPS BIOSCIENCE INC) for PARP2. To determine the IC50 value for the inhibitors, the compounds were tested using increasing concentrations of the test compounds and the average absorbance of each inhibitor concentration was plotted against the log of the concentration of inhibitor using the GraphPad Prism 6 program. Subsequently, the IC50s for the PARP1 or PARP2 inhibition were determined after non-linear fit using GraphPad Prism. The inhibitory effects of both the free base form (AMXI-5001-FB) and the HCl salt form (AMXI-5001-HCl) of AMXI-5001 were evaluated. DMSO was used as a negative control. Clinically approved PARP inhibitors Olaparib, Talazoparib, Niraparib, or Rucaparib were used as a positive control for PARP1 inhibition. Paclitaxel was also used as a negative control. (E) AMXI-5001 reduced PAR levels in situ in MDA-MB-436 human breast cancer cells: Cells were grown for 24 hr in media with or without varying concentrations of AMXI-5001, Olaparib, or Talazoparib. Cell lysates were prepared and the PAR levels were assessed by HT PARP In Vivo Pharmacodynamic Assay. The normalized ratio of pg PAR/ug total protein for cells treated with increasing doses of test compounds (AMXI-5001, Olaparib, or Talazoparib) over normalized ratio for DMSO Control-treated cells, were plotted against the compound concentrations using non-linear fit using GraphPad Prism. (F) AMXI-5001 profoundly inhibited the PAR levels in situ in MDA-MB-436 human breast cancer cells. Cells were grown for 4 hr in media with or without varying concentrations of AMXI-5001, Olaparib, or Talazoparib. Cell lysates were analyzed by western blot with an anti-PAR or anti-actin antibodies. AMXI-5001 inhibited cellular PAR expression in a comparable manner as the clinically approved PARPi. DMSO-treated (DMSO CTL) or untreated cells (Untreated CTL) cells were used as negative controls. (G) AMXI-5001 treatment induced a strong and dose dependent chromatin binding of both PARP1, and to a lesser extent PARP2: MDA-MB-436 cell lines were co-treated for 3 hr with 0.01% MMS and vehicle controls or increasing doses of AMXI-5001. Subsequently, cell lysates were prepared and fractionated into nuclear-soluble and chromatin-bound fractions then analyzed by western blot with an anti-PARP1 or anti-PARP2. Anti-H3 and anti TOP1 antibodies were used as control loading for chromatin- bound or soluble nuclear fractions, respectively.

Figure 2

AMXI-5001 is a potent microtubule polymerization inhibitor. Tubulin polymerization assay using the fluorescence-based tubulin polymerization assay (BK011P). A. Tubulin was incubated alone (Control), with Paclitaxel or Vinblastine. Each condition was tested in duplicate. Polymerization was measured by excitation at 360 nm and emission at 460 nm. The three Phases of tubulin polymerization are marked for the control (DMSO and Water) polymerization curve; I: nucleation, II: growth, III: steady state equilibrium. Data were plotted using the GraphPad Prism 6 program. B. AMXI-5001 and Vinblastine reduced the nucleation phase, and decreased the Vmax in dose dependent manner. Tubulin was incubated alone (DMSO Control), or in the presence of inhibitors (AMXI-5001 or Vinblastine), at increasing doses ranging from 0.16 μM to 5 μM. Each condition was tested in duplicate. C. Equipotent microtubule polymerization inhibition between free base (FB) and HCl- salt forms of AMXI-5001. Tubulin was incubated alone (DMSO Control), or in the presence of inhibitors either free base form or HCl salt form of AMXI-5001 at increasing doses ranging from 0.3125 μM to 10 μM. Each condition was tested in duplicate.

Figure 3

Comparaison of the anti-microtubule effect for AMXI-5001 versus standard microtubule targeting agents and PARP inhibitors. A549 cells were treated with either AMXI-5001, Vinblastine, Paclitaxel, Olaparib or Talazoparib at 0.05 μM, 0.5 μM, or 5 μM for 24 h. 0.1% DMSO or PARP inhibitors (Olaparib and Talazoparib) were used as a negative control. Vinblastine was used as a positive control for tubulin polymerization inhibition. Paclitaxel was used as a positive control for tubulin polymerization enhancement. Samples were then prepared as in the “Experimental” section, and the status of microtubules was observed using a Zeiss AxioImager 2 microscope; 63x objective, DAPI and RFP channels. Microtubule filaments are stained in red, and cell nuclei are stained in blue. AMXI-5001 inhibited the polymerization of microtubules in comparable manner as Vinblastine. PARP inhibitors does not affect the status of cellular microtubule polymerization when compared to controls.

Figure 4

A. Effect of AMXI-5001 on MDA-MB-436 Xenograft Growth and Body Weight in Female Athymic Nude Mice (31-Day Dosing). A. MDA-MB-436 xenografts tumors were established by inoculation of female athymic nude mice with 3.5 × 10⁶ MDA-MB-436 cells subcutaneously in the third mammary fat. When the tumors reached approximately ~100 mm³, mice were randomized to five treatment groups (8 animals per group): 1) Vehicle control administered BID orally on 5 day ON and 2 day OFF cycles, or AMXI-5001 administered at 2) 10 and 3) 50 mg/kg orally BID on 5 day ON and 2 day OFF cycles, or 4) Olaparib at 50 mg/k BID on 5 day ON and 2 day OFF cycles, or 5) vinblastine at IP 1 mg/kg once a week. All animals were treated over a 31 day dosing period. Tumor size for each animal was measured twice a week. B. Body weights were recorded twice a week for animals in each group. C. Histologic therapy response. Histological analysis using H&E staining at low (10 ×) and high (20 ×) magnifications of representative paraffin embedded MDA-MB-436 xenograft tumor sections form mice treated with: vehicle control, AMXI-5001 at 10 mg/kg or 50 mg/kg BID, Olaparib at 50 mg/kg BID 5 day on 2 day off cycles, vinblastine at 1 mg/kg once a week. All treatments were administered over 31 days dosing period.

Figure 5

Effect of AMXI-5001 on Large MDA-MB-436 Xenograft Growth and Body Weight in Female Athymic Nude Mice. A. AMXI-5001 causes uniformly growth inhibition and complete regression without recurrence of large MDA-MB-436 mammary fat xenograft tumors in all treated mice. Five mice bearing large tumors (Tumor size ranging from ~554 to ~1318 mm³) in each group. B. Body weights were recorded twice a week for animals in each group. No significant effect on the body weight during the entire course of treatment with AMXI-5001 at 50 mg/kg as compared to vehicle control treated group was observed.

Figure 6

Effect of AMXI-5001, Olaparib, Paclitaxel, and Olaparib/Paclitaxel on MDA-MB-436 Xenograft Growth and Body Weight in Female Athymic Nude Mice (56-Day Dosing). MDA-MB-436 xenografts tumors were established by inoculation of female athymic nude mice with 3.5 × 10⁶ MDA-MB-436 cells subcutaneously in the third mammary fat. When the tumor size average reached approximately ~200 mm³, mice were randomized to five treatment groups (8 animals/group): vehicle control, administered BID orally on 5 day ON and 2 day OFF cycles, AMXI-5001 (50 mg/kg) orally BID on 5 day ON and 2 day OFF cycles, Olaparib (50 mg/kg) BID on 5 day ON and 2 day OFF cycles, paclitaxel IV (30 mg/kg) once a week, or a combination therapy of Olaparib (50 mg/kg PO BID 5 day ON 2 day OFF) with paclitaxel (30 mg/kg) IV once per week. A. Tumor size for each animal was measured twice a week. B. Body weights were recorded twice a week for animals in each group.