ALK2 Inhibition Via TP-0184 Abrogates Inflammation-Induced Hepcidin Expression and Is a Potential Therapeutic for Anemia of Chronic Disease

Abstract

Hepcidin, a key liver peptide hormone, is essential to the regulation of bioavailable iron and erythropoiesis. Activin-like kinase receptor 2 (ALK2) signaling, via SMAD transcription factors, plays an important part in the regulation of hepcidin expression induced by pro-inflammatory cytokines. In chronic inflammatory conditions, such as rheumatoid arthritis, chronic kidney disease, colitis, and in some forms of cancer, hepcidin expression is induced. This induction of hepcidin expression results in lower levels of bioavailable iron, ultimately leading to the onset of anemia. Hepcidin regulates bioavailable iron levels by binding to and inhibiting the cellular iron pump, ferroportin. Ferroportin is important to macrophage-based iron recycling and dietary iron absorption. Several reports have suggested that lowering hepcidin provides a novel approach for targeting the clinical challenge of anemia. Currently approved approaches for these patients rely on transfusions and the use of erythropoietin-based therapies. Unfortunately, neither of these approaches address the underlying chronic inflammation or hepcidin induction and resulting anemia. In this report, we validate our small molecule inhibitor of ALK2, TP-0184, for the treatment of hepcidin-driven anemia of chronic diseases. Biochemical assays demonstrate that TP-0184 inhibits of the kinase activity of ALK2 with an IC50 of 5 nM. In vitro, TP-0184 is effective at targeting hepcidin expression with an EC50 lower than 100 nM in HepG2 cells. Three in vivo models were also explored for our validation of TP-0184. In our first study, turpentine oil TO was injected into the intrascapular fat pad of C57BL/6 mice to induce an acute inflammatory response that results in hepcidin-driven anemia. The animals were dosed with TP-0184 1 hour prior to TO treatment and once again 8 hours later. The TO-mediated acute inflammatory response in mice resulted in a 14-fold increase in liver hepcidin levels. Two oral doses of TP-0184 at 100 mg/kg, separated by 8 hours, reversed the induction of hepcidin that followed TO treatment. TP-0184 was tested at multiple doses in which efficacy was observed. In our second in vivo model, we induced anemia via intraperitoneal injection with heat-inactivated Brucella abortus. The mice were treated daily with TP-0184 for 3-7 days, after which, whole blood, plasma and livers were collected, from which liver and plasma hepcidin, plasma iron, and complete blood counts were assessed. Treatment with 100 mg/kg TP-0184 completely abrogated the Brucella abortus-induced reduction of hemoglobin and total red blood cell counts. In our third in vivo study, TP-0184 was also evaluated in the TC-1 lung cancer model for cancer-induced anemia. TC-1 tumor-bearing animals exhibited a 3-fold increase in liver hepcidin levels, which was reversed by dosing with 25 mg/kg TP-0184. From these experiments, we conclude that TP-0184 is a potent and selective inhibitor of ALK2 with demonstrated activity in multiple preclinical models of anemia associated with inflammation. TP-0184 also demonstrates favorable pharmacokinetic properties as well as good drug-like qualities, making it a strong candidate molecule with which to move into IND-enabling studies and formal clinical development. The current study supports a clinical development approach focused on anemia of chronic disease where an erythropoietin-sparing approach might offer significant clinical benefit to patients. Disclosures Peterson: Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

Role of hepcidin‐ferroportin axis in the pathophysiology, diagnosis, and treatment of anemia of chronic inflammation

“…However, because dorsomorphin and its derivatives do not specifically target BMP/SMAD signaling to hepcidin, they are considered suboptimal candidates for therapeutic development. TP‐0184, a specific ALK2 inhibitor (Toledo Pharmaceuticals), has recently been shown to suppress hepcidin in mouse models of ACI and cancer, but further studies are required to evaluate its full translational potential.…”

Therapeutic Advances in Regulating the Hepcidin/Ferroportin Axis

“…Asshoff et al recently developed the antibody momelotinib that targets ALK2 leading to a reduction in hepcidin production in anaemic rats [98]. Lastly, TP-0184 demonstrated an inhibitory effect against ALK2 with two oral doses eight hours apart ameliorating turpentine oil mediated anaemia in mice [99]. While both momelotinib and TP-0184 appear to be specific for only ALK2, both dorsomorphin and LDN-1913189 have been found to have off target effects, which reduces their potential as a therapeutic agents [132].…”

Hepcidin Therapeutics

“…The drug is currently undergoing a phase 1 clinical trial to determine its maximum tolerated dose and dose-limiting toxicities in patients with advanced solid tumors (ClinicalTrials.gov Identifier: NCT03429218). In preclinical mouse models of inflammation- and cancer-induced anemia, TP-0184 decreased hepatic hepcidin mRNA and improved hemoglobinization [117,118].…”