The Nitric Oxide-Generating Prodrug Js-K Is Selectively Toxic Towards AML Compared to Normal Human Hematopoietic Cells
Authors: Smock Kristi J. , Heaton William L. , Pomicter Anthony D. , Crist Ronda A , Kosak Ken M , Boucher Kenneth M , Rodgers George M. , O'Hare Thomas , Deininger Michael W. , Shami Paul J 
Published: October 2019

Abstract
Nitric oxide (NO) induces apoptosis and differentiation in acute myeloid leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a lead agent of the arylated diazeniumdiolate class. JS-K is active in vitro and in vivo against AML, multiple myeloma, and several solid tumors. JS-K is directly cytotoxic to malignant cells and inhibits angiogenesis in vitro and in vivo. We have previously shown that JS-K is not toxic towards normal murine hematopoietic cells.
Aiming at its clinical application to treat AML, we have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic®P123 polymers. The formulation (P123/DMSO) consists of 2.25% P123 and 2% dimethyl sulfoxide in phosphate buffered saline. P123/JS-K is currently at an advanced stage of pre-clinical development.
Here, we compared the cytotoxicity of JS-K and its formulation between normal hematopoietic and AML cells. HL-60 AML cells or normal human CD34+ cells isolated from 2 different cord blood units were treated for 24 hours with P123/DMSO or P123/JS-K before plating in semi-solid media. Colonies were scored at 10 days. P123/DMSO alone had no effect on colony formation by HL-60 or normal CD34+ cells. P123/JS-K at concentrations of 0.1, 0.25, and 0.5 μM inhibited HL-60 colony growth by 27, 74, and 100%, respectively (P < 0.05 for the comparison between 0.25, 0.5 μM and controls).
At the same concentrations, P123/JS-K inhibited the growth of erythroid colonies from the first cord blood sample, by 26, 43, and 61%, respectively. At a concentration of 0.1 μM, P123/JS-K did not inhibit the growth of erythroid colonies from the second cord blood sample but inhibited erythroid colony growth by 18, and 27% at concentrations of 0.25 and 0.5 μM, respectively. However, the differences with untreated controls were not statistically significant for either cord blood sample.
At the same concentrations P123/JS-K inhibited the growth of myeloid colonies from the first cord blood sample, by 8, 3, and 37%, respectively. At a concentration of 0.1 μM, P123/JS-K did not inhibit the growth of myeloid colonies from the second cord blood sample but inhibited myeloid colony growth by 2, and 6% at concentrations of 0.25 and 0.5 μM, respectively. However, the differences with untreated controls were not statistically significant for either cord blood sample.
With clinical development in mind, we also sought to determine whether the P123/DMSO formulation affects platelet function. P123/DMSO was added to normal donor blood obtained in either EDTA or citrate anticoagulant. With either anticoagulant, P123/DMSO did not affect the measured hemoglobin, white blood cell, or platelet counts. Inspection of peripheral blood smears (Wright stain) obtained with either anticoagulant revealed no platelet clumping. We then studied the effect of P123/DMSO on platelet aggregation in vitro in response to ADP, collagen, ristocetin, or arachidonic acid. Light transmission platelet aggregometry was performed using platelet-rich plasma from a healthy volunteer with normal platelet counts and function. In order to determine whether P123/DMSO could enhance or inhibit platelet aggregation, we chose a range of agonist concentrations from sub-threshold to concentrations expected to induce complete aggregation. P123/DMSO was added at a concentration expected to be equal to peak in vivolevels based on a prior dog toxicology study. Normal aggregation responses were observed in the saline control. There were no significant differences between this control and responses observed in the presence of P123/DMSO. Specifically, significantly decreased aggregation in P123/DMSO-treated platelets was not observed in response to agonists expected to elicit complete aggregation responses. Likewise, enhanced aggregation was not observed in response to sub-threshold agonist concentrations.
Our results confirm the potent anti-leukemic activity and limited toxicity against normal human hematopoietic cells of JS-K formulated in P123 micelles. Furthermore, the formulation does not affect platelet aggregation. We therefore expect that P123/JS-K will have a favorable hematologic toxicity profile and as such, will constitute an important addition to our armamentarium for the treatment of AML.

Disclosures
Deininger: Gilead: Research Funding; BMS: Consultancy, Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Shami:JSK Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.