Frank Schwoebel

The effects of the anti-hepcidin Spiegelmer NOX-H94 on inflammation-induced anemia in cynomolgus monkeys

Anemia of chronic inflammation is the most prevalent form of anemia in hospitalized patients. A hallmark of this disease is the intracellular sequestration of iron. This is a consequence of hepcidin-induced internalization and subsequent degradation of ferroportin, the hepcidin receptor and only known iron-export protein. This study describes the characterization of novel anti-hepcidin compound NOX-H94, a structured L-oligoribonucleotide that binds human hepcidin with high affinity (Kd = 0.65 ± 0.06 nmol/L). In J774A.1 macrophages, NOX-H94 blocked hepcidin-induced ferroportin degradation and ferritin expression (half maximal inhibitory concentration = 19.8 ± 4.6 nmol/L). In an acute cynomolgus monkey model of interleukin 6 (IL-6)-induced hypoferremia, NOX-H94 inhibited serum iron reduction completely. In a subchronic model of IL-6-induced anemia, NOX-H94 inhibited the decrease in hemoglobin concentration. We conclude that NOX-H94 protects ferroportin from hepcidin-induced degradation. Therefore, this pharmacologic approach may represent an interesting treatment option for patients suffering from anemia of chronic inflammation.

Effect of the antihepcidin Spiegelmer lexaptepid on inflammation-induced decrease in serum iron in humans

Increased hepcidin production is key to the development of anemia of inflammation. We investigated whether lexaptepid, an antihepcidin l-oligoribonucleotide, prevents the decrease in serum iron during experimental human endotoxemia. This randomized, double-blind, placebo-controlled trial was carried out in 24 healthy males. At T = 0 hours, 2 ng/kg Escherichia coli lipopolysaccharide was intravenously administered, followed by an intravenous injection of 1.2 mg/kg lexaptepid or placebo at T = 0.5 hours. The lipopolysaccharide-induced inflammatory response was similar in subjects treated with lexaptepid or placebo regarding clinical and biochemical parameters. At T = 9 hours, serum iron had increased by 15.9 ± 9.8 µmol/L from baseline in lexaptepid-treated subjects compared with a decrease of 8.3 ± 9.0 µmol/L in controls (P < .0001). This study delivers proof of concept that lexaptepid achieves clinically relevant hepcidin inhibition enabling investigations in the treatment of anemia of inflammation. This trial was registered at www.clinicaltrial.gov as #NCT01522794.

Hematopoietic stem and progenitor cell mobilization in mice and humans by a first-in-class mirror-image oligonucleotide inhibitor of CXCL12.

NOX‐A12 is a PEGylated mirror‐image oligonucleotide (a so‐called Spiegelmer) that binds to CXCL12 (stromal cell–derived factor‐1, SDF‐1) with high affinity thereby inhibiting CXCL12 signaling on both its receptors, CXCR4 and CXCR7. In animals, NOX‐A12 mobilized white blood cells (WBCs) and hematopoietic stem and progenitor cells (HSCs) into peripheral blood (PB). In healthy volunteers, single doses of NOX‐A12 had a benign safety profile and also dose‐dependently mobilized WBCs and HSCs into PB. HSC peak mobilization reached a plateau at five times the baseline level at an i.v. dose of 5.4 mg/kg. In accordance with the plasma half‐life of 38 h, the duration of the WBC and HSC mobilization was long lasting and increased dose‐dependently to more than 4 days at the highest dose (10.8 mg/kg). In conclusion, NOX‐A12 may be appropriate for therapeutic use in and beyond mobilization of HSCs, e.g., in long‐lasting mobilization and chemosensitization of hematological cancer cells.

Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.

The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.

Safety, pharmacokinetics and pharmacodynamics of the anti‐hepcidin Spiegelmer lexaptepid pegol in healthy subjects

Anaemia of chronic disease is characterized by impaired erythropoiesis due to functional iron deficiency, often caused by excessive hepcidin. Lexaptepid pegol, a pegylated structured l‐oligoribonucleotide, binds and inactivates hepcidin.

Abstract 3847: The anti-hepcidin Spiegelmer® Lexaptepid Pegol (NOX-H94) as treatment of anemia of chronic disease in patients with multiple myeloma, low grade lymphoma, and CLL: A phase II pilot study

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lexaptepid pegol (LP) is a PEGylated L-stereoisomer RNA aptamer that binds and neutralizes hepcidin. Hepcidin, a 25 amino acid peptide induced by inflammatory stimuli is a pivotal regulator of iron resorption and iron release from intracellular stores, which are severely impaired in anemia of chronic disease. Disturbances in iron metabolism resulting in functional iron deficiency are a key component of anemia of chronic disease which frequently complicates malignant disease. We evaluated the pharmacokinetics, pharmacodynamics, safety and efficacy of hepcidin blockade by LP as sole treatment of anemia of chronic disease in patients with multiple myeloma, low grade Non-, or Hodgkin lymphoma. Twelve patients with functional iron deficiency anemia with the following baseline characteristics, presented as median (range), were enrolled: age 64 years (35-77), Hb 9.6 g/dL (8.0-10.7), serum ferritin 317 µg/L (193-2805), serum iron 29 µg/dL (18-97), TSAT 12% (6-46). LP was injected i.v. at a dose of 1.2 mg/mg, TIW for 4 weeks. Blood counts, serum biochemistry, and iron status were evaluated weekly until two weeks post treatment and at week four after the end of therapy. The primary endpoint was increase in Hb by ≥1 g/dL at any time between start of therapy until 1 week after end of treatment. The study has the clinicaltrials.gov identifier NCT 01691040. Five of the 12 patients reached the target Hb increase of ≥1 g/dL, 3 patients achieved this goal within 2 weeks. Four of the 5 responding patients had hypochromic anemia (MCH 22-26 pg) and moderately increased baseline ferritin levels (200-350 µg/L). Median serum ferritin decreased from 317 to 232 µg/L (p=0.014) in the entire cohort of patients, and from 253 to 203 µg/L in responders (p=n.s). Reticulocyte hemoglobin increased from 22.0 to 25.2 pg (p=0.019) in responding patients, while in non-responders no increase was noted (30.0 to 30.1 pg). Similarly, a trend to increased reticulocyte index was observed in the responding patients (median: 0.9 to 1.3, p=n.s.) only. Soluble transferrin receptor levels (sTFR) showed a trend to decrease in responders (10.6 to 10.3 mg/L, p=n.s.), but remained unchanged in non-responders. Treatment with LP was well tolerated without major adverse reactions. In conclusion, LP induced a significant increase in Hb levels (≥1 g/dL) in 5 of 12 patients. Responding patients showed an increase in red cell and reticulocyte hemoglobin, and decrease in sTFR. These results support the concept of hepcidin inhibition for treatment of cancer associated anemia with functional iron deficiency. Patients with signs of iron deficiency as documented by hypochromic anemia, no excessive increase in ferritin and high sTFR levels showed a higher likelihood to respond to LP. Citation Format: Pencho Georgiev, Mihaela Lazaroiu, Luminita Ocroteala, Janet Grudeva-Popova, Emanuil Gheorghita, Mariana Vasilica, Sanda M Popescu, Andrei Cucuianu, Luciana Summo, Frank Schwoebel, Kai Riecke, Heinz Ludwig. The anti-hepcidin Spiegelmer® Lexaptepid Pegol (NOX-H94) as treatment of anemia of chronic disease in patients with multiple myeloma, low grade lymphoma, and CLL: A phase II pilot study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3847. doi:10.1158/1538-7445.AM2014-3847