Preclinical Evaluation of CRx-501, a Potent Selective A2A Agonist as a Novel Drug Candidate for the Treatment of Multiple Myeloma
Authors: Rickles Richard J , Pierce Laura , Giordano Thomas , Avery William , Farwell Melissa , Crowe David , Tam Winnie F. , Chen Mei , Kansra Vikram , McMillin Douglas W. , Anderson Kenneth C , Mitsiades Constantine , Lee Margaret S. 
Published: April 2020

Abstract
We have made the surprising discovery that agonism of adenosine A2A receptors has potent and synergistic antiproliferative effects in B-cell malignancies. Here we describe the preclinical evaluation of CRx-501, a potent selective adenosine A2A receptor agonist that synergizes with established anti-MM agents resulting in enhanced efficacy in pre-clinical models of MM. CRx-501 demonstrates potent single agent inhibition of proliferation in MM cell lines with EC50s ranging from 2–20 nM and maximum effects ranging from 20–75% inhibition of proliferation in the 10 MM cell lines surveyed. While CRx-501 demonstrates single agent effects, this molecule potently synergizes with glucocorticoids (dexamethasone and prednisolone), bortezomib, lenalidomide, melphalan and doxorubicin as well as emerging drug classes including HDAC inhibitors and HSP90 inhibitors. Substantial increases in overall effect levels and 2 to 100 fold potency shifts are observed with CRx-501 combinations in a broad panel of 10 MM cell lines including those both sensitive and resistant to current MM agents. In MM.1S cells, addition of 50 nM CRx-501 to 100 nM Dexamethasone results in 95% inhibition of proliferation as compared to approximately 60 and 75% inhibition with either CRx-501 or dexamethasone alone respectively. This combination results in a 10 fold shift in the dexamethasone IC50 and a combination index of 0.2 indicating high levels of synergy. Importantly, in cells resistant to dexamethasone up to the highest concentrations tested (2 microM; EJM, ANBL-6, MM.1R, KSM-12PE, MOLP-8), addition of CRx-501 can induce synergistic inhibition of proliferation converting resistance into sensitivity. Similarly, in combination with lenalidomide, CRx-501 results in a &gt;50 fold shift in IC50, an enhancement of efficacy from 50% to 90% inhibition of proliferation and activity against a lenolidamide resistant cell line (MOLP-8). Synergy is also observed with bortezomib resulting in a 2-fold shift in the IC50 of bortezomib in the presence of CRx-501. Importantly, CRx-501 is highly selective for B-cell malignancies and demonstrates no single agent activity or synergy in normal primary human cell types including peripheral blood mononuclear cells, umbilical vein endothelial cells, aortic smooth muscle cells, coronary artery endothelial cells and solid tumor cell lines at concentrations 2–3 orders of magnitude greater than the IC50 in MM cell lines. These antiproliferative effects occur through apoptosis. Treatment with CRx-501 in combination with dexamethasone causes a rapid and synergistic induction of Annexin V expression as compared to either single agent alone. Surprisingly, the potency and efficacy of CRx-501 is enhanced in the presence of 10 ng/mL interleukin-6 and HS-5 human bone marrow stromal cells. Concentrations of CRx-501 that are effective in vitro are achievable in mice with a 3 mg/kg s.c. dose resulting in a mean plasma AUC0–24 of 1228 ng*hr/mL. The synergistic antiproliferative effects of CRx-501 translate into xenograft models of MM with no significant body weight loss. Mice bearing subcutaneous H929 tumors show a 73% reduction in tumor volume after treatment with the combination of dexamethasone (1 mg/kg, s.c QD) and CRx-501 (3 mg/kg s.c. QD) for 34 days compared to a 36% or 37% decrease after treatment with either CRx-501or dexamethasone respectively. In summary, we report the preclinical evaluation of CRx-501, a potent and highly synergistic A2A agonist as a novel, selective, synergistic drug candidate for the treatment of MM. Our preclinical data provides compelling evidence in support of the further development of CRx-501 for use in multi-drug combination therapy of MM.