Werner Purschke

Spiegelmer Inhibition of CCL2/MCP-1 Ameliorates Lupus Nephritis in MRL-(Fas)lpr Mice

The monocyte chemoattractant protein CCL2 is crucial for monocyte and T cell recruitment from the vascular to the extravascular compartment at sites of inflammation. CCL2 is expressed in human lupus nephritis and was shown to mediate experimental lupus; therefore, CCL2 antagonists may be beneficial for therapy. This study describes the l-enantiomeric RNA oligonucleotide mNOX-E36, a so-called Spiegelmer that binds murine CCL2 with high affinity and neutralizes its action in vitro and in vivo. The mirror image configuration of the Spiegelmer confers nuclease resistance and thus excellent biostability. mNOX-E36 does not induce type I IFN via Toll-like receptor-7 or cytosolic RNA receptors, as recently shown for certain synthetic D-RNA. Autoimmune-prone MRL(lpr/lpr) mice that were treated with a polyethylene glycol form of mNOX-E36 from weeks 14 to 24 of age showed prolonged survival associated with a robust improvement of lupus nephritis, peribronchial inflammation, and lupus-like inflammatory skin lesions. Thus, mNOX-E36-based inhibition of CCL2 represents a novel strategy for the treatment of autoimmune tissue injury, such as lupus nephritis.

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.

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.

A novel C5a-neutralizing mirror-image (l-)aptamer prevents organ failure and improves survival in experimental sepsis.

Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.

A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes

Background: An increased glucagon/insulin ratio is known to contribute to hyperglycemia in diabetes. Results: NOX-G15, a mirror-image mixed DNA/RNA glucagon-neutralizing aptamer, was identified. It improved glucose tolerance in models of type 1 and 2 diabetes. Conclusion: NOX-G15 may be useful for treatment of type 1 and 2 diabetes. Significance: The new therapeutic candidate may help to reduce insulin need in diabetes. Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2′-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.

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.

Polyetheylenimine-Polyplexes of Spiegelmer NOX-A50 Directed against Intracellular High Mobility Group Protein A1 (HMGA1) Reduce Tumor Growth in Vivo

High mobility group A1 (HMGA1) proteins belong to a group of architectural transcription factors that are overexpressed in a range of human malignancies, including pancreatic adenocarcinoma. They promote anchorage-independent growth and epithelial-mesenchymal transition and are therefore suggested as potential therapeutic targets. Employing in vitro selection techniques against a chosen fragment of HMGA1, we have generated biostable l-RNA oligonucleotides, so-called Spiegelmers, that specifically bind HMGA1b with low nanomolar affinity. We demonstrate that the best binding Spiegelmers, NOX-A50 and NOX-f33, compete HMGA1b from binding to its natural binding partner, AT-rich double-stranded DNA. We describe a formulation method based on polyplex formation with branched polyethylenimine for efficient delivery of polyethylene glycol-modified Spiegelmers and show improved tissue distribution and persistence in mice. In a xenograft mouse study using the pancreatic cancer cell line PSN-1, subcutaneous administration of 2 mg/kg per day NOX-A50 formulated in polyplexes showed an enhanced delivery of NOX-A50 to the tumor and a significant reduction of tumor volume. Our results demonstrate that intracellular targets can be successfully addressed with a Spiegelmer using polyethylenimine-based delivery and underline the importance of HMGA1 as a therapeutic target in pancreatic cancer.

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.

A novel CGRP‐neutralizing Spiegelmer attenuates neurogenic plasma protein extravasation

Calcitonin gene‐related peptide (CGRP) plays an important role in the pathology of migraine, and recent clinical trials suggest the inhibition of CGRP‐mediated processes as a new therapeutic option in migraine. In this study, we describe the generation of NOX‐L41, a CGRP‐neutralizing mirror‐image (l‐)aptamer (Spiegelmer) and investigate its in vitro and in vivo function.

The Glutamine Synthetase from the Hyperthermoacidophilic Crenarcheon Sulfolobus acidocaldarius: Isolation, Characterization and Sequencing of the Gene

The glutamine synthetase (EC 6.3.1.2) from the hyperthermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was purified to homogeneity, characterized and the glnA gene isolated and sequenced. The amount of enzyme present in the cytosolic fraction from Sulfolobus cells showed a strong variation depending on the carbon and nitrogen sources in the growth medium. The enzyme was found to be a dodecameric protein composed of identical subunits of 52 kDa. It was stable at 78 degrees C in the presence of Mn2+ ions. The catalytic activity was regulated solely by feed-back inhibition through L-alanine and glycine and not by adenylylation. No evidence for the presence of isoenzymes was found. Sequence comparison showed that the Sulfolobus protein is most closely related to the glutamine synthetases of the I-beta type despite its regulatory properties and the finding that the known euryarcheal glutamine synthetase sequences belong to the I-alpha subgroup of these enzymes. Our phylogenetic analysis suggests that the gene duplication leading to the development of the I-alpha and I-beta enzymes preceded the separation of the archea and the bacteria.