Stefan Zöllner

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.

SDF-1 Inhibition Targets the Bone Marrow Niche for Cancer Therapy

Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells.

Blockade of SDF-1 after irradiation inhibits tumor recurrences of autochthonous brain tumors in rats.

Background Tumor irradiation blocks local angiogenesis, forcing any recurrent tumor to form new vessels from circulating cells. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas in the brains of nude mice can be delayed or prevented by inhibiting circulating blood vessel–forming cells by blocking the interaction of CXCR4 with its ligand stromal cell-derived factor (SDF)–1 (CXCL12). In the present study we test this strategy by directly neutralizing SDF-1 in a clinically relevant model using autochthonous brain tumors in immune competent hosts. Methods We used NOX-A12, an l-enantiomeric RNA oligonucleotide that binds and inhibits SDF-1 with high affinity. We tested the effect of this inhibitor on the response to irradiation of brain tumors in rat induced by n-ethyl-N-nitrosourea. Results Rats treated in utero with N-ethyl-N-nitrosourea began to die of brain tumors from approximately 120 days of age. We delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age, began treatment with NOX-A12 immediately following irradiation, and continued with either 5 or 20 mg/kg for 4 or 8 weeks, doses and times equivalent to well-tolerated human exposures. We found a marked prolongation of rat life span that was dependent on both drug dose and duration of treatment. In addition we treated tumors only when they were visible by MRI and demonstrated complete regression of the tumors that was not achieved by irradiation alone or with the addition of temozolomide. Conclusions Inhibition of SDF-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme.

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.

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 385: Inhibition of recurrences of experimental brain tumors after irradiation by blocking the activity of SDF-1 (CXCL12) using the Spiegelmer® NOX-A12.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC With current treatment of glioblastomas 85% of the tumors recur within the field of high dose radiation. This tumor recurrence requires formation of new blood vessels. We have developed a new therapeutic paradigm based on the dual origin of tumor blood vessels: Angiogenesis - the sprouting of endothelial cells from nearby blood vessels, and vasculogenesis - the formation of blood vessels from circulating cells. Because tumor irradiation abrogates local angiogenesis the tumor must rely on the vasculogenesis pathway for regrowth after irradiation. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas implanted intracranially in the mouse brain can be delayed or prevented by inhibiting circulating blood vessel forming cells by blocking the interaction of the chemokine receptor CXCR4 with its ligand SDF-1 (CXCL12)1. However, SDF-1 has a second receptor, CXCR7, which has been implicated in endothelial cell migration, is present on tumor vasculature, and is potentially also able to activate vasculogenesis. To block both receptors we have therefore used NOX-A12, an L-enantiomeric RNA oligonucleotide (Spiegelmer®), which inhibits SDF-1 with subnanomolar affinity. We have tested the effect of this inhibitor on the response to irradiation of the U251 human GBM in nude mice and on ENU-induced brain tumors in the Sprague-Dawley rat. In the mouse model we found that inhibition of SDF-1 post-irradiation was highly effective in enhancing tumor response to a single dose of 20 Gy whole brain irradiation. In the rat model the pups from ENU-treated pregnant mothers begin to die of brain tumors from approximately 120 days of age. We performed two studies with this model: In the first we delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age and began treatment with NOX-A12 immediately following irradiation and continued every 2 days with either 5 or 20 mg/kg injected subcutaneously for either 4 or 8 weeks. These doses and times were chosen as equivalent to human doses and times that based on existing data have been found to be safe and well tolerated in human volunteers and which are effective in inhibiting the action of SDF-1. We found a marked prolongation of life of the rats that was dependent both on drug dose and length of treatment. In the second study we measured the size of the tumors post-irradiation using MRI and again found a pronounced enhancement of the efficacy of irradiation that was superior to the addition of temozolomide at doses equivalent to those used with concomitant radiotherapy in treating GBM. We believe that these encouraging data justify a human trial in first line glioblastoma patients. 1. Kioi M, Vogel H, Schultz G, Hoffman RM, Harsh GR, Brown JM: Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice. J Clin Invest 120:694-705, 2010 Citation Format: Shie-Chau Liu, Reem Alomran, Jason Stafford, Milton Merchant, Taichang Jang, Stefan Zollner, Anna Kruschinski, Laurence Recht, J. Martin Brown. Inhibition of recurrences of experimental brain tumors after irradiation by blocking the activity of SDF-1 (CXCL12) using the Spiegelmer® NOX-A12. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 385. doi:10.1158/1538-7445.AM2013-385