PARP1/2; tubulin/microtubule; tubulin polymerization

AMXI-5001 potently and selectively inhibits PARP1 and PARP2. AMXI-5001 binds to the catalytic domain of human PARP1.AMXI-5001 is a week tankyrase inhibitor. AMXI-5001 is a potent tubulin polymerization inhibitor.AMXI-5001 has no significant off target effect. AMXI-5001 targets tumor cells with or without defects in homologous recombination. AMXI-5001 effect on cellular checkpoint, regulatory, and signaling proteins. Synergistic anticancer activity of AMXI-5001 with approved anticancer therapies in vitro [1].

Initial 5-day study in MDA-MB-436 xenografts [1]
AMXI-5001 (HCl salt form) was formulated in 10% TPGS and administered orally to female athymic nude mice bearing established MDA-MB-436 xenograft tumors, an aggressive basal breast carcinoma cell line cell line that harbors BRCA1 deletion and is BRCA1 deficient. When tumors reached average volume of 350 mm3, AMXI-5001 suspension was administered orally at doses of 12.5, 25, or 50 mg/kg/dose BID for 5 days. Five days of twice per day (BID) oral administration of AMXI-5001 caused a rapid and marked tumor growth inhibition at doses of 25 and 50 mg/kg/dose BID, and clear tumor regression at 50 mg/kg/dose BID compared to the vehicle group (Figure S32A).
Moreover, plasma and tumor tissue bioanalyses revealed a dose dependent increase in AMXI-5001 plasma concentration and a corresponding dose dependent increase in AMXI-5001 tumor concentration (Figure S32B). However, there was no significant changes in AMXI-5001 concentration in either plasma or tumor tissues between tissues harvested after 4th and 10th repeat oral dose (Figure S32B). These results suggest that there is no significant increase in the accumulation of AMXI-5001 in either blood or tumor tissue with repeat treatment.
Western Blot analyses of tumor lysates showed that AMXI-5001 modulates its targets in vivo in a dose dependent manner (Figure S33). Particularly, AMXI-5001 inhibition of its targets was more evident at 25 and 50 mg/kg BID doses as indicated by a noticeable decrease in polymeric adenosine diphosphate (ADP) ribose (PAR) expression, a surrogate marker for PARP activity, and in total tubulin expression, a surrogate marked for microtubule polymerization inhibition (Figure S33). This provides support for the hypothesis that the anti-tumor efficacy of AMXI-5001 potentially results from inhibition of both PARP and microtubule polymerization. In general, there was a good correlation between increases in plasma and tumor drug concentrations and increased inhibition of either or both of these AMXI-5001 targets at the doses tested.

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Confirmatory 31-day study in MDA-MB-436 xenografts [1]
The efficacy of AMXI-5001 seen in the 5-day study was confirmed in this subsequent study of a longer dosing duration. 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, oral; 2) 10 mg/kg PO BID AMXI-5001 on 5-day ON/2-day OFF cycles, oral; 3) 50 mg/kg PO BID AMXI-5001 on 5-day ON/2-day OFF cycles, oral; 4) 50 mg/kg BID Olaparib on 5-day ON/2-day OFF cycles, oral; 5) 1 mg/kg vinblastine once weekly (Q1W), intraperitoneally. All animals were treated over a 31-day dosing period. Tumor size for each animal was measured twice a week (Figure 4A).
AMXI-5001 exhibited significant, dose-dependent tumor growth inhibition. In addition to tumor growth inhibition, AMXI-5001 also exhibited clear tumor regression in this model. For the 50-mg/kg AMXI-5001 group, all tumors experienced a complete regression. By Day 31 of the dosing schedule, tumors were either too small to be accurately measured or non-palpable. Comparison to Olaparib revealed that the anti-tumor growth effect observed with AMXI-5001 at a dose of either 10 or 50 mg/kg/dose BID was superior to Olaparib at a dose of 50 mg/kg/dose BID. In contrast to AMXI-5001, Olaparib did not exhibit any tumor regression activity in this model and vinblastine given at a dose of 1 mg/kg Q1W showed no significant effect on tumor growth in this model (Figure 4A).
Animals treated with AMXI-5001 doses of 10 and 50 mg/kg/dose BID on 5-day ON/2-day OFF cycles for 31 days, did not display any physical symptoms of compound-related toxicity, nor did Olaparib and vinblastine treated animals. There was no significant effect on the body weight during the entire course of treatment in any group (Figure 4B).
In order to assess treatment related histological changes, paraffin sections of harvested tumors taken at the end of treatment were stained with hematoxylin-eosin (H&E). H&E staining revealed dramatic hemorrhagic tumor necrosis in tumors treated with AMXI-5001 at 50 mg/kg dose (Figure 4C). The harvested tumors from the mice treated with AMXI-5001 at 50 mg/kg were nonpalpable by the end of the treatment. Tumor sections from this group showed a few remaining xenograft cancer cells dispersed through extensive fibrosis, which were surrounded by skin, skeletal muscle and fat from the host animal. At higher magnification a photograph of these tumors showed a significant treatment effect with evidence of necrosis and hemorrhage, with hemosiderin filled macrophage accumulation, cholesterol granuloma, chronic inflammation and fibrosis (Figure 4C). The tumors from mice treated with AMXI-5001 at 10 mg/kg also showed reduced density of xenograft cells with areas of necrosis, inflammation, and fibrosis. Tumor sections from vehicle-treated, Olaparib-treated or vinblastine-treated mice, contained mostly vital tumor cells without any fibrosis (Figure 4C).
At the end of the study, tumors were resected and processed for analysis of microtubule filament formation. Sections from paraffin embedded tumors were stained with an antibody specific to alpha/beta-tubulin, and visualized by fluorescent microscopy. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Repeat oral dose of AMXI-5001 at either 10 mg/kg or 50 mg/kg BID resulted in a striking dose dependent inhibition of microtubule filament formation in MDA-MB-436 cells derived tumors (Figure S34). Vinblastine treatment also induced a marked inhibition of microtubule filament in MDA-MB-436 tumors. Conversely, repeat oral treatment with Olaparib at 50 mg/kg BID dose showed no effect on tumor cells microtubule filaments formation.
Pharmacodynamic parameters for AMXI-5001 treatment, were also determined using lysate prepared with xenograft tumor specimens from each group in this study. For PARP inhibition, PAR levels were evaluated, and for microtubule destabilization, total alpha/beta tubulin expression levels were assessed by western blot analyses with their specific corresponding antibodies. AMXI-5001 treatment at either 10 or 50 mg/kg inhibited dramatically, and in a dose dependent manner, PAR expression in MDA-MB-436 derived tumors (Figure S35). AMXI-5001 treatment also resulted in a significant and dose dependent decrease in the expression levels of total alpha/beta-tubulin in MDA-MB-436 derived tumors (Figure S35). Olaparib treatment resulted in a significant inhibition of PAR expression but had no effect on tubulin expression in MDA-MB-436 derived tumors. In contrast, vinblastine treatment showed no effect on levels of PAR or total tubulin expression in MDA-MB-436 derived tumors (Figure S35). Consistent with PARP inhibition effect, both AMXI-5001 treatment and Olaparib treatment resulted in a conspicuous inhibition of the tumor protein expression of the cell cycle checkpoint protein (CHFR), as compared to vehicle control treatment (Figure S35).
Collectively, the aforementioned results, indicate that AMXI-5001 modulates in a dose dependent manner, both its intended targets in vivo in tumors. These results also suggest that antitumor activity of AMXI-5001 might be attributed to its dual mechanism of action through its synchronous inhibition of both PARP and microtubule polymerization in the tumor cells.

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Complete regression of established large tumors (600-1300 mm3) in MDA-MB-436 xenografts with single agent AMXI-5001 treatment [1]
Based on the anti-tumor efficacy in the above study in MDA-MB-436 xenografts when tumors were staged at 100-150 mm3 before initiation of dosing, the anti-tumor effect of AMXI-5001 was tested in larger, more well-established tumors (Figure 5A).
At the end of the 31-day dosing period of the above described study, 5 out of 8 mice from the control group with tumor sizes of 554-1318 mm3 received AMXI-5001 at a dose of 50 mg/kg/dose BID on 5-day ON/2-day OFF cycles, 2 mice from the vinblastine group, and 1 mouse from the control group (tumor size 458-953 mm3) received vehicle control on the same schedule. Remarkably, all large tumors treated with AMXI-5001 exhibited gradual and approximatively complete tumor regression starting in the first week of treatment initiation (Figure 5A). Notably, following AMXI-5001 treatment, there was no sign of tumor recurrence 2 weeks after mice stopped treatment (Figure 5A). Conversely, in the new vehicle treated group, tumors continued to grow rapidly and the mice were terminated, less than three weeks after vehicle treatment was initiated, because of excessive tumor growth and the consequent poor animal health. There was no significant effect on the body weight during the entire course of treatment with AMXI-5001 at 50 mg/kg/dose BID as compared to vehicle control treated group (Figure 5B).

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Superior antitumor effect of AMXI-5001 compared to combination therapy of single-agent PARP and microtubule inhibitors in MDA-MB-436 xenograft model [1]
The objective of this study was to compare the in vivo anti-tumor efficacy of AMXI-5001 treatment to either single agent Olaparib (a clinical PARP inhibitor), or single agent paclitaxel (a potent clinical microtubule polymerization inhibitor), and to combination therapy of Olaparib and paclitaxel in MDA-MB-436 Xenograft model. Olaparib and paclitaxel were administered at clinically relevant doses and schedules.
AMXI-5001 induced complete or near-complete tumor regression in all treated animals, and this effect was superior to either single agent Olaparib or paclitaxel, or combination treatment with both agents (Figure 6A). In this tumor model, single agent paclitaxel treatment displayed weak anti-tumor activity, whereas treatment with single agent Olaparib induced a clear but modest inhibition of the tumor growth. Combination therapy of Olaparib and paclitaxel resulted in a greater tumor growth inhibition than either of these single agents alone. However, the combination therapy of Olaparib and paclitaxel failed to induce tumor regression and the tumors continued to grow, albeit, at slower rate than the vehicle treated or single agent (Olaparib or paclitaxel) treated tumors. AMXI-5001 was much more effective in this model than the combination of single agent Olaparib with single agent paclitaxel.
Animals treated with AMXI-5001, single agent Olaparib, or combination treatment of Olaparib with paclitaxel did not show any notable treatment-related toxicity (Figure 6B). However, 3 out of 8 mice in the single-agent paclitaxel group showed lethargy, lack of response to external stimuli, loss of consciousness, and labored respiration within a few minutes after first intravenous (IV) administration of paclitaxel (30 mg/kg). The 3 affected animals in this group were euthanized. Nevertheless, all remaining 5 animals in the single agent paclitaxel treated group did not show any notable treatment-related toxicity for the duration of treatment. There were no significant treatment-related effects on body weight during the entire course of treatment with any of the treatment modalities as compared to the vehicle control treatment.

In vitro kinase inhibition assays [1]
The potential for AMXI-5001 to inhibit off target kinases was assessed in a panel of 156 recombinant human kinase activity and binding assays including cytoplasmic and receptor tyrosine kinases, serine/threonine kinases and lipid kinases. The kinase profiling assays were performed using Life Technologies’ SelectScreen® Profiling Service, with a broad coverage of the human kinome. The kinase activity assays measure peptide phosphorylation or ADP production while the kinase binding assays monitor displacement of ATP site-binding probes. The ATP concentrations used in the activity assays were within 2-fold of the experimentally determined apparent Michaelis constant (K m app) value for each kinase while the competitive binding tracer concentrations used in the binding assays were within 3-fold of the experimentally determined dissociation constant (K d) values. AMXI-5001 was tested at 8 µM in triplicate against each kinase and the mean % inhibition values are reported. For selected kinases, 10-point inhibitor titrations were carried out using the same kinase assays as used in the single point tests in order to determine the inhibitor concentration providing 50% inhibition (IC50).

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PARP2 inhibition assay [1]
The inhibitory action of the test compounds towards PARP2 was determined using a commercially available microplate assay kit and in accordance with the instructions provided by the manufacturer. Subsequently, the IC50s for the PARP2 inhibition were determined after non-linear fit using GraphPad Prism.

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PARP enzyme assays [1]
The inhibitory action of the test compounds towards PARP1 was determined using a commercially available microplate assay kit and in accordance with the instructions provided by the manufacturer. Briefly, stock solutions of the various test compounds were made in dimethyl sulfoxide (DMSO). For the assay, each strip well was filled with 10 µL of the inhibitor solution, 20 μL of diluted PARP1 enzyme (providing 0.5 Unit/well), and 25 µL of PARP Cocktail (consisting of biotinylated NAD, activated DNA in Tris-Cl pH 8.0, and EDTA). The strip wells were incubated at room temperature for 60 min, and then washed 4 times with phosphate buffered saline (PBS: Na2HPO4, NaH2PO4, and NaCl) and 0.1% Triton X-100, and 2 times with PBS. Then, 50 µL of diluted Strep-HRP (blocking solution) was added to each well, and the strips were further incubated at room temperature for 60 min. After washing the wells 2 times each with PBS and with 0.1% Triton X-100, and 2 times with PBS they were mixed with 50 µL of TACS-Sapphire™ colorimetric substrate, and allowed to stand in the dark for 15 min. After stopping the reaction by adding 50 µL of 0.2 N hydrochloric acid (HCl) to each well, the absorbance was measured at 450 nm. Parallel experiments were conducted by substituting the test solution with an equivalent volume of DMSO to verify the effect of the vehicle on the enzyme activity. All the assays were conducted in at least two separate occasions, each time in duplicates Subsequently, the IC50s for the PARP1 inhibition were determined after non-linear fit using GraphPad Prism. The results of these studies are reported in μM. DMSO was used as a negative control. Clinically approved PAPR inhibitors (olaparib, talazoparib, niraparib, or rucaparib) were used as a positive control for PARP inhibition. In some instances, paclitaxel was also used as a negative control.

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In vitro tubulin polymerization assay [1]
An in vitro fluorescence-based tubulin polymerization assay kit was used according to the manufacturer’s protocol for monitoring the time-dependent polymerization of tubulin to microtubules. The reaction mixture, containing porcine brain tubulin, fluorescent reporter and GTP in presence or absence of test compounds, was prepared. Tubulin polymerization was followed by monitoring fluorescence enhancement due to the incorporation of a fluorescent reporter into microtubules as polymerization proceeded. Fluorescence emission (excitation at 360 nm and elimination at 460 nm wavelength) was measured for one hour and 45 minutes at one min intervals in a Biotek Synergy Plate Reader. Under the conditions described above, approximately 45% of the tubulin is polymerized, leaving flexibility for detecting enhancers and inhibitors of polymerization. DMSO was used as a negative control. Paclitaxel was used as a positive control for tubulin polymerization enhancement. Vinblastine was used as a positive control for tubulin polymerization inhibition. The incorporation of the fluorescent reporter into microtubules as polymerization proceeded was measured. The fluorescence absorbance, at 360 nm excitation and 460 nm elimination wavelengths, was plotted against the reaction time using the GraphPad Prism 6 program. The Vmax were determined after non-linear fit using GraphPad Prism. The Vmax of DMSO control was set to 100% polymerization The Vmax of the DMSO control was set to 100% polymerization. The percentages of compounds Vmax over DMSO control Vmax were plotted against the compound concentrations. Subsequently, The IC50s for the tubulin polymerization inhibitors were determined after non-linear fit using GraphPad Prism.

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Competitive MS binding assay [1]
To determine the binding site of AMXI-5001 on tubulin, competitive MS binding assay was conducted as previously described but with slightly different conditions for Colchicine, vinblastine and paclitaxel binding. This assay was used to assess the tubulin binding of Colchicine, Vinblastine and Paclitaxel and to identify which of these three binding sites that AMXI-5001 binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the flow through fraction can be accurately determined using highly sensitive and specific HPLC-MS/MS method. Briefly, Nocodazole Colchicine, and Vinblastine were incubated with tubulin in the incubation buffer at 37°C for 1 h. Paclitaxel was incubated with performed tubulin in the incubation buffer at 37°C for 1 hr. Performed tubulin was prepared by incubating tubulin with GTP in the incubation buffer at 37°C for 1 hr. Varying concentrations of Nocodazole, vincristine and Docetaxel were used to compete with Colchicine-, Vinblastine- and Paclitaxel-tubulin binding, respectively. After incubation, reaction samples were centrifuged in an Ultracel-30 microconcentrators. The flow through (unbound ligands) was collected and analyzed by HPLC MS/MS, as described below. Varying concentrations of AMXI-5001 were examined to individually compete with colchicine-, vinblastine- and paclitaxel-tubulin binding. The ability of the competitor or AMXI-5001 to inhibit the binding of ligands was expressed as a percentage of unbound ligand control in the absence of any competitor.

Cellular PAR western blot assay [1]
The effect of AMXI-5001 on the cellular PAR levels was also assessed by standard western blotting procedures. Briefly, cancer cells were cultured overnight in 6 wells plates, followed by incubation with vehicle control or test compound for 24 h. The cells were then washed and cell lysates were prepared for western blotting as described below. Protein concentration in each cell lysate was quantified using BCA method. Immunoblotting was subsequently performed using standard procedures. A total of 10 μg of protein was resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and probed with Anti-PAR and secondary antibodies. Clinically approved PARPi (olaparib, talazoparib, or rucaparib) were used as a positive control for PAR inhibition and γ H2AX induction. β-actin was used as the loading control.

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Cellular PARP-DNA trapping assays [1]
To assess the PARP trapping ability of AMXI-5001, standard cellular trapping assays were performed as previously described. Briefly, cancer cells were cultured overnight in 6 wells plates, followed by cotreatment with alkylating agent methyl methanesulfonate (MMS) and vehicle control, or cotreatment with alkylating agents and varying concentrations of the test PARPi for 1 or 3 hr. Cells were then washed and collected by trypsinization. Subsequently chromatin fractions were prepared using Thermo Scientific subcellular protein fractionation kits per manufacturer protocol. Samples were normalized for protein concentration and analyzed by immunoblotting by anti-PARP1, anti-TOP1, and anti-H3. Clinically approved PARPi (olaparib, talazoparib, or rucaparib) were used as a positive control for PARP-DNA trapping. To quantify the PARPi-induced PARP1-DNA trapping in human cancer cells, densitometry was performed on immunoblots. DNA-bound PARP1 levels were normalized to total cellular PARP1 levels (chromatin-bound PARP1+ Unbound PARP1). Each experiment was conducted at least three independent times. Representative results are depicted in the result section below.

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Cell titer Glo assay [1]
Antiproliferative activity of AMXI-5001 was evaluated in a panel of 110 cancer cell lines of various origins, either proficient or defective for BRCA-1 or BRCA-2 expression or expressing mutant forms of the two genes. Proliferation was assessed after cells were exposed for 3 days or 6 days with AMXI-5001 doses ranging from 8 nM to 5 μM. Briefly, 5000 or 1000 cells (In 3 days or 6 days exposure, respectively) were cultured in a 96-well plate and incubated at 37°C and 5% CO2 prior to adding serial dilutions of AMXI-5001. Cells were then incubated for 3-6 days at 37°C and 5% CO2. Cell viability was assessed by Cell Titer Glo assay. The number of living cells was determined by reading the plate on a GloMax Luminametor. Cell growth was expressed as percentage growth with respect to vehicle (DMSO control) treated cells. The concentration required to inhibit cell growth by 50% (IC50) was determined after non-linear fit using GraphPad Prism. DMSO treated cells were used as a vehicle control. MTAs (Paclitaxel and Vinblastine) as well as clinical PARPis (Olaparib, Talazoparib, Niraparib, and Rucaparib) were used as comparative controls.

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Clonogenic assay [1]
The effect of AMXI-5001 on cell viability was also evaluated using colony formation assay in cells lines of ovarian, non-small cell lung cancer, and prostate origin. In brief, cell lines were incubated in 0.1% (v/v) DMSO (vehicle control) or 0.008, 0.04, 0.2, 1, 5 mM AMXI-5001 at 37°C until > 50 colonies have formed (6-28 days). Seeding cell density was determined from preliminary experiments and is defined as seeding density producing linear cell growth after cell incubation in 0.1% (v/v) DMSO (vehicle control). Cell viability was determined using the colony formation assay by counting Crystal Violet-stained colonies using ImageJ software. IC50 values were defined as the AMXI-5001 concentrations producing 50% cell viability when compared to vehicle control cells (100% cell viability). Clinical PARPis (Olaparib, Talazoparib, Niraparib, and Rucaparib) were used as comparative controls.

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Wound healing assay/scratch assay [1]
The ability of AMXI-5001 to inhibit cell migration was tested using in vitro scratch assay that assess cell migration by recovery of the scratch wound in A549 lung cancer cells grew into monolayer. Briefly, A549 lung cancer cells were seeded onto 12-well plates. When the cell confluence reached about 80% and above, scratch wounds were made by scraping the cell layer across each culture plate using the tip of 200 μl pipette. After wounding, the debris was removed by washing the cells with PBS. Wounded cultures were incubated in culture medium alone (Untreated control), or medium containing 0.1% (v/v) DMSO (DMSO control) or 0.04, 0.2, 1 mM AMXI-5001 at 37°C for 24 hr. Subsequently, 3 fields (40 Å~) were randomly picked from each scratch wound and visualized by microscopy to assess cell migration ability. Clinical PARPis (Olaparib and Talazoparib) and clinical MTAs (Paclitaxel and Vinblastine) were used as positive controls for cell migration inhibition.

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Cell cycle analysis [1]
The effect of AMXI-5001 on cell-cycle progression in dividing MDA-MB-436, OVCAR8 and A549 cells, was evaluated. Briefly, the cells were grown in T75 flasks and treated with 0.1% DMSO (vehicle control), Vinblastine, or AMXI-5001 at varying concentrations for 24 Hr. After incubation, the cells were trypsinized, washed with PBS and fixed. After fixation, the cells were centrifuged, washed with once with PBS and stained with propidium iodide. Finally, the distributions of treated cells at different phases of cell cycle (G1, S, G2/M) were analyzed using flow cytometry. The percentage of cells in each mitotic phase pre- and post-treatment with AMXI-5001 for 24 Hr was determined. MTAs (Paclitaxel and Vinblastine) were used as positive controls for cell cycle arrest.

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Western blot analysis of cellular checkpoint and signaling proteins [1]
To better understand the mechanisms by which AMXI-5001 regulates the cell cycle arrest in cancer cells, standard western analysis was performed on cell lysates to assess the status of various checkpoint-related proteins and cell signaling proteins (Figure 3). Briefly, cancer cells were cultured overnight in 6 wells plates in serum free medium, followed by incubation with vehicle control or test compound for 24 h. The cells were then washed and cell lysates were prepared for western blotting as described below. Protein concentration in each cell lysate was quantified using BCA method. Immunoblotting was subsequently performed using standard procedures. a total of 10 μg of protein was resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and probed with checkpoint proteins corresponding antibodies, and secondary antibodies. Clinically approved PARPi (Olaparib, Talazoparib) or MTAs (Paclitaxel, Vinblastine) were used as controls. β-actin and GAPDH were used as the loading control.

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Flow cytometry analysis-cell surface marker staining [1]
A549 cells were cultured overnight in 100 mm culture dishes, followed by incubation with vehicle control or AMXI-5001 at 1 mM or 5 mM for either 24 hr or 48 hr then harvested by trypsinization, as described below in method section. For PDL1 and death receptors (DR4 and DR5) cell surface staining, harvested cells were washed with PBS, and stained with respective antibodies in cell staining buffer and processed for flow cytometry analysis. BD CellQuest Pro software was used for flow cytometric data analysis.

Pharmacokinetic evaluations of AMXI-5001 in mice[1]
The objective of these studies was to determine pharmacokinetic and bioavailability profile following a single dose oral administration of either free base form or HCl salt forms of AMXI-500 in BALB/c mice. The In-Life procedures for these studies were conducted using Explora Bioloabs contract research services. The in vitro bioanalyses of the plasma samples were performed using Integrated Analytical Solutions contract research services. Briefly, AMXI-5001 free base and HCl salt form were formulated either as NMP/CMC suspension or in 10% TPGS suspension, respectively and administered orally to female BALB/c mice. The formulations protocols used for the PK studies are described below. The bioavailability for AMXI-5001 free base form was assessed following a single oral dose administration at either 50 or 100 mg/kg per mouse. The bioavailability for AMXI-5001 HCL form was assessed following a single oral dose administration at 50 mg/kg per mouse. Blood samples were collected at pre-dose, 0.5, 1, 2, 4, 8 and 24 hours after AMXI-5001 single dose oral administration. For each mouse, one-two time points were assigned. Each group had 3 blood samples per time point. The first blood collection was survival bleed and the second blood collection was terminal. Blood samples were collected into tubes containing K2EDTA anticoagulant and stored on wet ice until centrifuged and processed for plasma. Plasmas were stored at -80°C until analysis. The peak concentration (Cmax), the time to maximum concentration (Tmax), the half-life, and the AUC were determined from composite mean plasma concentration-time data. All doses and plasma concentrations of AMXI-5001 were presented as free base.

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Pharmacodynamic evaluations of AMXI-5001 in mice bearing MDA-MB-436 tumors[1]
The primary objective of this study was to determine the relationship between AMXI-5001 dose, plasma concentration, tumor concentration and inhibition of PARP and microtubule polymerization In vivo in MDA-MB-436 Xenograft Tumors. The In-Life procedures for this study were conducted using Explora Bioloabs contract research services. The in vitro bioanalyses of the plasma samples were performed using Integrated Analytical Solutions contract research services. Briefly, AMXI-5001 HCL salt form was formulated in 10% TPGS suspension, as described below and administered orally to female athymic nude mice bearing established MDA-MB-436 Xenograft Tumors (When tumors reach average volume of 500 mm3). AMXI-5001 suspension was administered orally BID at 6.25, or 12.5, or 25 mg/kg per mouse for 5 days. At 3 hr post the 4th and 10th dose, n=3 mice per group, or each AMXI-5001-dose and the vehicle control group, were euthanized and blood collected in EDTA tubes for plasma processing. At necropsy the tumor tissues from all groups, as well as stomach, small intestine, large intestine, caecum, kidneys, and liver from the vehicle control group and the group treated with AMXI-5001 at 50 mg/kg BID, were individually collected, placed into cryotubes and flash frozen in liquid nitrogen. The frozen plasma and tissue samples were kept at -80°C until bioanalysis for AMXI-5001 concentration. Plasma samples were analyzed by high performance liquid chromatography (HPLLC) in conjunction with a triple quadrupole mass spectrometer that used electrospray ionization in tandem with positive ionization (MS/MS). The lower limit of quantitation (LLOQ) was 5 ng/ml. The quantitative range of the assay was 5 to 10000 ng/ml.

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In vivo xenograft models[1]
In the present studies, the MDA-MB-436 (triple negative human breast carcinoma with BRCA1 mutation) was utilized to explore the efficacy and potency of AMXI-5001 with regard to tumor growth inhibition and regression in vivo. The In-Life procedures for these studies were conducted using the Explora Biolabs contract research services. The standard experimental design for these studies involved twice daily (BID) daily oral administration (PO) of AMXI-5001, following a 5-day ON and 2-day OFF cycles, beginning when the established solid tumors were staged (~100 -150 mm3 for most xenografts, ~700 mm3 and up to 1500 mm3 for xenograft regression studies). Throughout the dosing period of 10-60 days, tumor size and body weight were measured twice weekly.
At the end of the study, tumors were resected and processed for analysis of microtubule filament formation. Sections from paraffin embedded tumors were stained with an antibody specific to for alpha/beta-tubulin, and visualized by fluorescent microscopy.
In order to assess treatment related histological changes, paraffin sections of harvested tumors taken at the end of treatment were also stained with hematoxylin-eosin (H&E) and analyzed by a certified pathologist.
Pharmacodynamic parameters for AMXI-5001 treatment, were also determined using lysate prepared with xenograft tumor specimens from each group in this study. For poly {adenosine diphosphate (ADP)}-ribose polymerase (PARP) inhibition (PARPi), Poly (ADP-ribose) (PAR) levels were evaluated, and for microtubule destabilization, total alpha/beta tubulin expression levels were assessed by Western blot analyses with their specific corresponding antibodies.

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HT PARP in vivo pharmacodynamic assay[1]
The HT PARP In Vivo Pharmacodynamic Assay is a high-throughput, chemiluminescent ELISA designed to quantify poly (ADP-ribose) (PAR) in cellular extracts. The assay employs a two-site sandwich technique in which two different anti-PAR antibodies are used to capture and detect the target analyte. This assay is useful for measuring PAR in extracts from peripheral blood mononuclear cells (PBMC), cultured cells, and tissues. Additionally, this assay can be used to monitor the efficacy of PARPi or anti-cancer drugs on cellular PAR formation and cancer cell cytotoxicity. The inhibitory effect of AMXI-5001 on cellular PAR formation was quantified with this HT PARP In Vivo Pharmacodynamic Assay in accordance with the instructions provided by the manufacturer. Each ELISA plate contains serial dilutions of purified PAR standard used to plot the PAR standard curve. The net mean RLU (Relative Light Units) values of the PAR standards were calculated by subtracting the background (without PAR) from the RLU values, then plotted as a function of the corresponding PAR values (pg/ml). The PAR standard curve was plotted using the GraphPad Prism 6 program. Typically, the linear dynamic range for the PAR standard curve is from 10 to 1000 pg/ml. The net RLU values for each cell lysate sample was calculated by subtracting the background from the RLU values. Subsequently, the PAR levels in each sample was determined using the PAR standard curve. The PAR level of DMSO-treated control was set to 100% PAR level. The percentages of test compounds-treated samples over DMSO-treated control PAR levels were plotted against the compound concentrations. Subsequently, the IC50s for the cellular PAR formation inhibitors were determined after non-linear fit using GraphPad Prism.

Metabolism and PK properties of AMXI-5001 [1]
Currently, there are no approved orally bioavailable microtubule targeting agent. One of the objectives of our dual PARP and microtubule inhibitor discovery program was to develop an orally bioavailable dual PARP and microtubule inhibitor and to improve metabolic stability, PK properties and oral bioavailability over existing PARP1/2 inhibitors. In vitro metabolism studies of AMXI-5001 in hepatocytes from rats, dogs, cynomolgus monkeys, and humans demonstrated that AMXI-5001 had excellent liver stability; The half-life of AMXI-5001 (2 μM) during incubation with hepatocytes (500,000 cells/mL) from Sprague-Dawley rats, beagle dogs, cynomolgus monkeys, or humans was estimated to be 217 min, 812 min, 185 min, and 417 min, respectively. After incubation 1 μM concentration for 120 min at 37°C, the percentage of AMXI-5001 remaining was 63.6%, 85.4%, 64.1%, and 78.5% for rat, dog, monkey, and human hepatocytes, respectively (Not shown). These data suggest AMXI-5001 will have human clearance approximately similar to that of the animal species.

AMXI-5001 has been given orally to rats and dogs at various dose levels (manuscript in preparation). AMXI-5001 was absorbed and bioavailable in all species tested. In rats and dogs, exposure increased with the increase in dose level, supporting the use of these species in the toxicity studies of AMXI-5001. A variety of formulations were tested, eventually leading to selection of 10% TPGS (D-α-tocopherol polyethylene glycol-1000-succinate; Vitamin E) in 0.01 N HCl, pH 2.1-2.3, because of its suitable toxicity profile and ability to deliver adequate AMXI-5001 systemic exposure. AMXI-5001 demonstrated an absolute bioavailability in rats and dogs of 31% and 64%, respectively with this formulation, and PK properties that would predict a human half-life that is sufficient to support a regimen of twice daily administration (manuscript in preparation).

In vitro studies assessing the potential for inhibition of human cytochrome P450 enzymes (CYP450s) showed that AMXI-5001 did not inhibit any of the major human hepatic CYP450 enzymes CYP1A2, CYP2B6, CYP2D6, and CYP3A4/5. There was weak (and not time-dependent) inhibition of CYP2C9, and CYP2C19 (Data not shown).

Overall, AMXI-5001 demonstrated excellent metabolic stability, oral bioavailability and PK properties.

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AMXI-5001 pharmacokinetic assessment [1]
To support the in vivo primary pharmacodynamic studies in xenograft models, pharmacokinetic parameters of AMXI-5001 were determined in female BALB/C mice. AMXI-5001 free base or hydrochloride salt forms were formulated either as N-methyl-pyrrolidine/carboxymethylcellulose (NMP/CMC) suspension or in 10% D-α-tocopherol polyethylene glycol-1000-succinate; Vitamin E (TPGS) suspension, respectively and administered orally. AMXI-5001 was absorbed and bioavailable in this strain of mice, enabling its testing in murine xenograft models with oral dosing (Table S18).

[1]. AMXI-5001, a novel dual parp1/2 and microtubule polymerization inhibitor for the treatment of human cancers. Am J Cancer Res. 2020;10(8):2649-2676.

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.[1]

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AMXI-5001 demonstrated a remarkable In vivo preclinical anti-tumor activity in BRCA mutated triple negative breast cancer (TNBC) model, a cancer with currently no effective therapy. Oral administration of single agent AMXI-5001 induced complete regression of established tumors, including exceedingly large tumors (Figures 4, 5 and 6). Most importantly, none of the AMXI-5001 treated mice had tumor re-growth until the end of the study, two weeks after AMXI-5001 dosing stopped. Furthermore, AMXI-5001 resulted in superior anti-tumor effects when compared to either single agent (PARP inhibitor (Olaparib) or microtubule targeting agent (Paclitaxel or Vinblastine)) or combination treatment with both agents which were given at clinically relevant doses (Figures 4A, 6A). Importantly, the AMXI-5001 anti-tumor effect was achieved with tolerable toxicity, evidence of PARP and microtubule inhibition in vivo in tumors and favorable PK properties that allow twice-a-day oral dosing in human patients. AMXI-5001 is the first in class small-molecule inhibitor reported to date that offers a continuous and synchronous PARP and microtubule polymerization inhibitions, and thus results in synthetic lethality, particularly in cancer cells vulnerable to DNA damage. The discovery and characterization of AMXI-5001 as an orally bioavailable dual PARP and microtubule polymerization inhibitor, provides a welcome addition to the oncology field and we believe the pharmacological properties of AMXI-5001 warrant further investigation, and its advancement into clinical studies in cancer patients. [1]

C25H20FN5O3

457.46

457.155

C, 65.64; H, 4.41; F, 4.15; N, 15.31; O, 10.49

2170491-77-5

AMXI-5001 hydrochloride

145426155

Off-white to light yellow solid powder

4.2

3

6

6

34

795

0

C1C=CC2C(CC3=CC(C4C=CC5NC(NC(=O)OCC)=NC=5C=4)=C(C=C3)F)=NNC(=O)C=2C=1

DCSUGHIXMFUMOQ-UHFFFAOYSA-N

InChI=1S/C25H20FN5O3/c1-2-34-25(33)29-24-27-20-10-8-15(13-22(20)28-24)18-11-14(7-9-19(18)26)12-21-16-5-3-4-6-17(16)23(32)31-30-21/h3-11,13H,2,12H2,1H3,(H,31,32)(H2,27,28,29,33)

ethyl N-[6-[2-fluoro-5-[(4-oxo-3H-phthalazin-1-yl)methyl]phenyl]-1H-benzimidazol-2-yl]carbamate

2170491-77-5; AMXI 5001; AMXI-5001; AMXI5001; ethyl N-[6-[2-fluoro-5-[(4-oxo-3H-phthalazin-1-yl)methyl]phenyl]-1H-benzimidazol-2-yl]carbamate; CHEMBL5222013; SCHEMBL21575709; Z5889958807

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~109.30 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)