Abstract
Introduction
Anemia of inflammation (AI) is a frequent complication of chronic infection, inflammatory diseases, heart failure, kidney disease, and cancer. Current therapies are directed at the treatment of the underlying disease, as well as parenteral iron supplementation and administration of erythropoiesis-stimulating agents. In AI, inflammatory cytokines, including interleukin 6 (IL6), induce hepatic synthesis of hepcidin, a hormone that reduces serum iron levels. Induction of hepatic hepcidin gene expression by IL6 also requires bone morphogenetic protein (BMP) signaling. Parenteral administration of BMP signaling inhibitors, including LDN-193189 (LDN, a small molecule inhibitor of BMP type I receptor kinases) can increase hemoglobin levels in rodent AI models. Although LDN is orally bioavailable, the ability of enterally-administered LDN to inhibit BMP signaling and ameliorate AI is not known.
Methods
We studied 10 week old C57bl/6 mice, fed a regular diet. To characterize the pharmacokinetic profile of LDN after oral administration, LDN (1 to 10 mg/kg in citrate buffer, pH 3.1) or vehicle was administered to mice by gavage. Additional mice received a single intraperitoneal injection of LDN (3 mg/kg in citrate buffer, pH 3.1). One and two hours later, mice were sacrificed, and plasma and liver were harvested. Plasma and hepatic LDN levels were measured by chromatography-tandem mass spectrometry. The ability of orally-administered LDN to inhibit hepatic BMP signaling was assessed by measuring phosphorylation of BMP-responsive Smad proteins (Smads 1 and 5) using immunoblot techniques and expression of genes encoding Id-1 (a BMP target gene) and hepcidin using qRT-PCR.
To determine whether orally administered LDN can increase hemoglobin (Hb) levels in a mouse AI model, we induced chronic inflammation in mice by subcutaneously administering turpentine (5 mL/kg) once a week for three weeks. As controls, mice received weekly subcutaneous injections of saline. Turpentine-challenged mice were treated daily with LDN (1 mg/kg) or vehicle by gavage. One week after the third turpentine injection, blood was collected for measurement of complete blood counts.
Results
One hour after an oral administration of LDN (1, 3, and 10 mg/kg), plasma LDN levels were 133±27, 187±46, and 496±105 ng/mL, respectively, and hepatic LDN levels were 267±94, 259±98, and 610±22 ng/g wet tissue weight. Similar plasma and hepatic LDN levels were detected 2 hours after oral administration. One hour after intraperitoneal administration of LDN (3 mg/kg), plasma and liver LDN concentrations were 176±179 ng/mL and 178±80 ng/g, respectively.
All three doses of orally-administered LDN markedly reduced hepatic levels of phosphorylated Smads 1 and 5 at one and two hours. Similarly, at one and two hours after oral administration of all three doses of LDN, hepatic Id-1 and hepcidin mRNA levels were decreased by at least 80% and 50%, respectively. Intraperitoneal and oral LDN administration were similarly effective at reducing hepatic Smads 1 and 5 phosphorylation and Id-1 and hepcidin gene expression.
In response to turpentine injections, mice developed sterile abscesses associated with a systemic inflammation. Hb levels were less in turpentine-challenged mice that were treated with vehicle than in saline-challenged mice (12.7±0.7 vs 14.9±0.7 g/dL; p<0.0001) and were associated with a reduction in mean corpuscular volume (MCV, 43.2±0.5 vs 45.2±0.8 fl; p<0.0001). Hb levels and MCVs were greater in turpentine-challenged mice that were treated with orally-administered LDN (13.6±0.5 g/dL and 44.1±0.5 fl, respectively) than in turpentine-challenged mice that were treated with vehicle (p<0.005 and p<0.006, respectively).
Conclusion
LDN is an orally bioavailable BMP type I receptor kinase inhibitor. Oral administration of LDN inhibits hepatic BMP signaling and hepcidin gene expression and can increase hemoglobin levels in a mouse AI model. LDN may represent a novel therapeutic approach to the treatment of AI that does not require parenteral administration.
Disclosures:
Off Label Use: LDN-193189, BMP type I receptor kinase inhibitor. Yu:MGH: Patents & Royalties. Bloch:MGH: Patents & Royalties.
Discovery of N-((4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): A Highly Potent, Selective, and Orally Bioavailable Inhibitor of TGF-β Type I Receptor Kinase as Cancer Immunotherapeutic/Antifibrotic Agent
