Effective Preclinical Management of Angiosarcoma With Oral PaclitaxelMurray J. Cutler1, Krista E. Belko1, Yahao Bu1, Alissa R. Verone-Boyle1, David Cutler1, Wing-Kai Chan1, Rudolf Kwan1, Johnson Y.N. Lau1, and Michael P. Smolinski1

1Athenex, Inc., Buffalo, NY •

INTRODUCTION • Angiosarcomas are a type of soft tissue sarcoma characterized by rapidly proliferating, extensively

infiltrating, anaplastic cells derived from blood and lymphatic vessels.1

• Angiosarcomas arise at various body sites, including in cutaneous tissue, in soft tissue, and at visceral locations. Angiosarcomas are frequently metastatic at diagnosis, with a natural history complicated by local recurrence, distant metastases, and poor overall survival.1,2

• Angiosarcomas represent about 2% of soft tissue sarcomas and 5.4% of cutaneous soft tissue sarcomas.3-5

• There is no US Food and Drug Administration (FDA)-approved treatment for angiosarcoma, and survival is limited with the therapies that are currently administered. However, reports in the literature of objective tumor response support the use of intravenous (IV) paclitaxel in the treatment of angiosarcoma.6-10

• However, IV paclitaxel therapy is commonly accompanied by hypersensitivity reactions and irreversible neuropathy.

• Oral paclitaxel and encequidar (formerly HM30181A), a highly selective, potent, non-absorbable P-glycoprotein inhibitor, eliminates the risk of hypersensitivity to IV formulations while potentially reducing neuropathy, with the potential for improved efficacy due to prolonged AUC.

• Oral paclitaxel and encequidar is currently being tested in a pivotal Phase III trial for the treatment of patients with metastatic breast cancer and in multiple Phase II trials for solid tumors. A study of oral paclitaxel in patients with cutaneous angiosarcoma is ongoing (NCT03544567).11

Figure 1. Oral Paclitaxel and Encequidar - A New Chemotherapy Paradigm

Improved tolerability allows for multiple doses

IV Paclitaxel Oral Paclitaxel and Encequidar

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Figure 2. Encequidar: An Oral, Highly Selective, Potent P-Glycoprotein Inhibitor

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METHODS

In Vitro Cytotoxicity of Paclitaxel in Angiosarcoma Cell LinesMS1, SVRA221a, SVRbag4, and human dermal microvascular endothelial (HMEC-1) cells were seeded in 96-well plates (1500 cells per well) in culture medium supplemented with 1 μM encequidar. After 24 hours’ incubation at 37°C, cells were washed once then treated with paclitaxel at a range of concentrations (0.1-2187 nM) in 100 μL of culture medium for 72 hours. Three independent experiments of 2 replicates were completed. A 10 μL aliquot of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution [5 mg/mL in phosphate-buffered saline (PBS)] was added to each well and incubated at 37°C for 2 hours. The culture medium was removed followed by addition of 100 μL of dimethyl sulfoxide to each well. The absorbance in each well was measured at 540 nm using a SpectraMax microplate reader (Molecular Devices, San Jose, CA).

A sigmoid Emax model using nonlinear regression [Equation (1)] with least squares fit was used to estimate Emax and GI50 values using GraphPad Prism 6 for each experiment:

(1) % inhibition =Emax∙ C γ

C γ + GI50

where Emax is the maximal effect on cell proliferation caused by drug treatment; GI50 is the drug concentration that causes 50% of the maximal growth inhibition; C is the concentration of drug; and γ is the Hill coefficient, which is a measure of steepness of slope. The average Emax and GI50 values and standard deviated from 3 individual experiments were calculated and reported. R-squared of all curve fits exceeded 0.9.

Figure 3. In Vitro Cytotoxicity of Paclitaxel in Murine Angiosarcoma Cell Lines.

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(A) MS1, (B) SVRA221a, and (C) SVRbag4 cells were treated with paclitaxel (0.1 - 2178 nM) for 72 hours. Symbols represent

the mean of each individual experimental observation and lines depict the fit of the sigmoid Emax pharmacodynamic model

using nonlinear regression. All data are representative of n = 3.

Figure 4. In Vitro Cytotoxicity of Paclitaxel in a Human Endothelium Cell Line.

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nonlinear regression. All data are representative of n = 3

Immunofluorescence Microscopy of Tubulin Disruption Coverslips (22 mm) were precoated with rat tail collagen for 1 hour then seeded with cells in 6-well plates for 24 hours before treatment. Murine angiosarcoma cells were treated with paclitaxel for 3 hours, followed by fixation with cold methanol at -200°C for 20 minutes. After blocking with 2% bovine serum albumin (BSA) in PBS for 30 minutes, cells were incubated with anti-a-tubulin antibody (Sigma T6199) diluted in blocking buffer (2% BSA in PBS) for 1.5 hours at room temperature. Cells were washed 3 times with PBS and incubated with Alexa 598 anti-Mouse IgG (Invitrogen, Carlsbad, CA) for 1 hour at room temperature. Following 3 PBS washes, coverslips were mounted on glass slides in ProLong antifade reagent (Invitrogen). Fluorescent images were captured using a Nikon TE2000-E inverted microscope for 2 independent experiments.

SVRA221a Subcutaneous Angiosarcoma Mouse ModelA preliminary study that focused on model development (M200-ATX010.101) determined the optimal inoculation conditions (tumor cell number and use of Matrigel) and evaluated percentage of engraftment, tumor doubling time, and time to reach the size endpoint of SVRA221a cells in severe combined immunodeficiency (SCID) mice. Tumor cells (1 x 106) in 50% Matrigel were inoculated into one shaved flank of female SCID mice. When the majority of the tumors reached a tumor volume of 150 mm3 to 200 mm3, mice were sorted into treatment groups such that the mean tumor volume across study groups was equivalent. Mice received 20 mg/kg oral (PO) encequidar followed 30 minutes later by 10 mg/kg, 20 mg/kg, 30 mg/kg, or 40 mg/kg PO paclitaxel. The treatment schedule was administered QDx3/week for 3 weeks. Tumors and body weights were measured 3 times per week for up to 4 weeks. Mice were euthanized at 4 weeks’ posttreatment initiation, when tumors reached 1500 mm3, or when they became necrotic or ulcerated.

Figure 5. Immunofluorescence Microscopy of Tubulin.

(A) MS1, (B) SVRA221a, and (C) SVRbag4 cells were treated with paclitaxel (1 - 100 nM) in the presence and absence of

100 nM encequidar for 3 hours. Representative photos of experimental data (n = 2).

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Figure 6. The Effect of Oral Paclitaxel and Encequidar on SVRA221a Tumor Growth in SCID Mice.

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Mice (n = 10) were treated with the indicated agents by gavage beginning when the majority of tumors were 150-200 mm3.

Dosing followed a QDx3/week schedule for up to 3 weeks. Orange arrows indicate treatment days. Encequidar or its vehicle

was administered 30 minutes before paclitaxel or its vehicle. Data are presented as the mean ± standard error of the mean

(n ≥ 7 tumors per data point).

RESULTSThe potency (IC50) of paclitaxel was estimated to be 1.8 ± 0.36 ng/mL for MS1, 1.8 ± 1.0 ng/mL for SVRA221a, 3.9 ± 1.0 ng/mL for SVRbag4, and 0.46 ± 0.23 ng/mL for HMEC-1 cells. Immunofluorescence was used to examine the effect of paclitaxel on microtubule polymerization in the angiosarcoma cell lines. Paclitaxel induced polymerization of microtubules accompanied with condensed and rounded morphology starting at 4.2 ng/mL to 8.5 ng/mL.

Table 1. Inhibition of angiosarcoma cell proliferation by treatment with paclitaxel.

SpeciesGI50

(ng/mL)Emax

(% inhibition)

MS1 Mouse 1.8 ± 0.36 93 ± 4.5

SVRA221a Mouse 1.8 ± 1.0 96 ± 1.1

SVRbag4 Mouse 3.9 ± 1.0 99 ± 1.4

HMEC-1* Human 0.46 ± 0.23 99 ± 0.10

Mean ± standard deviation , n = 3

*Human dermal microvascular endothelial cells

A sigmoid Emax pharmacodynamic model using nonlinear regression was used to estimate the GI50 and Emax of paclitaxel.

To assess the efficacy of oral paclitaxel, MS1, SVRA221a, and SVRbag4 xenograft models were developed. The MS1 tumors grew too slowly, whereas the SVRbag4 tumors did not reach the tumor size endpoint because of necrosis and ulceration. Therefore, tumor doubling time and tumor growth delay could not be determined. In the SVRA221a angiosarcoma xenograft model, oral paclitaxel administered for 3 consecutive days, weekly for 3 weeks, resulted in dose-dependent tumor growth inhibition and subsequent increased survival.

The most frequent cage side observations were related to changes in tumor appearance, including a darkened appearance with some bruising around the tumor and the development of necrotic or ulcerated areas. No significant weight loss was observed.

A dose-dependent decrease in SVRA221a tumor growth appeared to plateau between 30 mg/kg and 40 mg/kg of paclitaxel. Maximum growth inhibition (61.1%) was achieved on day 13 with 30 mg/kg paclitaxel.

Cavernous blood filled neoplastic vessels were consistently formed in vehicle and 10 mg/kg paclitaxel-treated groups. However, few to none were present in the 20 mg/kg, 30 mg/kg, or 40 mg/kg paclitaxel-treated groups, suggesting that oral paclitaxel demonstrated dose-dependent efficacy in this angiosarcoma murine model.

Figure 7. The Effect of Oral Paclitaxel and Encequidar on Survival.

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2 20 mg/kg Encequidar + 10 mg/kg Paclitaxel 16.5 0.136

3 20 mg/kg Encequidar + 20 mg/kg Paclitaxel 19.0 0.018

4 20 mg/kg Encequidar + 30 mg/kg Paclitaxel 25.0 0.002

5 20 mg/kg Encequidar + 40 mg/kg Paclitaxel 22.0 0.015

Mice (n = 14) were treated by gavage as indicated following a QDx3/week schedule for up to 3 weeks. Encequidar or its

vehicle was administered 30 minutes before paclitaxel or its vehicle. (A) Kaplan-Meier survival curves, (B) Median survival

time (MST), and significance determined by the log-rank test. P < 0.05 is significant.

Figure 8. Angiosarcoma Histopathology.Representative images of tumors from vehicle-treated (A & B) and 30 mg/kg oral paclitaxel-treated (C & D) mice.

Blood-filled cavernous neoplastic vessels/sinuses

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CONCLUSIONS • Oral paclitaxel administered 30 minutes after encequidar resulted in a dose-dependent

effect on SVRA221a tumor growth as demonstrated by both increased survival times and decreased tumor volumes. The antitumor effect of oral paclitaxel appeared to plateau between 30 mg/kg and 40 mg/kg, suggesting that increasing the dose above 30 mg/kg provides no additional benefit.

• Treatment-associated toxicity was not observed, as illustrated by Total Condition Scores of 10 to 12 and weight loss of <10% for the majority of the mice, regardless of treatment. Tumors appeared dark in color with some bruising, which is indicative of the vascular nature of angiosarcomas and often leads to ulceration and necrosis.

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Encequidar is a potent and highly selective non-ATP competitive, reversible P-glycoprotein (P-gp) inhibitor (new chemical entity) with minimal systemic absorption that exerts its activity in the intestinal epithelium

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Presented at the American Association for Cancer Research (AACR) Annual Meeting, March 29–April 3, 2019, Atlanta, GA, USA

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