Sven Klussmann

Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury

Objective Monocyte chemoattractant protein-1 (MCP-1, CCL2), the primary ligand for chemokine receptor C–C chemokine receptor 2 (CCR2), is increased in livers of patients with non-alcoholic steatohepatitis (NASH) and murine models of steatohepatitis and fibrosis. It was recently shown that monocyte/macrophage infiltration into the liver upon injury is critically regulated by the CCL2/CCR2 axis and is functionally important for perpetuating hepatic inflammation and fibrogenesis. The structured L-enantiomeric RNA oligonucleotide mNOX-E36 (a so-called Spiegelmer) potently binds and inhibits murine MCP-1. Pharmacological inhibition of MCP-1 with mNOX-E36 was investigated in two murine models of chronic liver diseases. Methods Pharmacological inhibition of MCP-1 by thrice-weekly mNOX-E36 subcutaneously was tested in murine models of acute or chronic carbon tetrachloride (CCl4)- and methionine–choline-deficient (MCD) diet-induced chronic hepatic injury in vivo. Results Antagonising MCP-1 by mNOX-E36 efficiently inhibited murine monocyte chemotaxis in vitro as well as migration of Gr1+ (Ly6C+) blood monocytes into the liver upon acute toxic injury in vivo. In murine models of CCl4- and MCD diet-induced hepatic injury, the infiltration of macrophages into the liver was significantly decreased in anti-MCP-1-treated mice as found by fluorescence-activated cell sorting (FACS) analysis and immunohistochemistry. In line with lower levels of intrahepatic macrophages, proinflammatory cytokines (tumour necrosis factor α, interferon γ and interleukin 6) were significantly reduced in liver tissue. Overall fibrosis progression over 6 (CCl4) or 8 weeks (MCD diet) was not significantly altered by anti-MCP-1 treatment. However, upon MCD diet challenge a lower level of fatty liver degeneration (histology score, Oil red O staining, hepatic triglyceride content, lipogenesis genes) was detected in mNOX-E36-treated animals. mNOX-E36 also ameliorated hepatic steatosis upon therapeutic administration. Conclusions These results demonstrate the successful pharmacological inhibition of hepatic monocyte/macrophage infiltration by blocking MCP-1 during chronic liver damage in two in vivo models. The associated ameliorated steatosis development suggests that inhibition of MCP-1 is an interesting novel approach for pharmacological treatment in liver inflammation and steatohepatitis.

In vivo properties of an anti-GnRH Spiegelmer: An example of an oligonucleotide-based therapeutic substance class

Spiegelmers are high-affinity l-enantiomeric oligonucleotide ligands that display high resistance to enzymatic degradation compared with d-oligonucleotides. The target binding properties of Spiegelmers can be designed by an in vitro-selection process starting from a random pool of oligonucleotides. Applying this method, a Spiegelmer with high affinity (KD = 20 nM) for the peptide hormone, gonadotropin-releasing hormone (GnRH) was isolated. The Spiegelmer acts as an antagonist to GnRH in Chinese hamster ovary cells stably expressing the human GnRH receptor, and its activity is unchanged by linking to 40-kDa polyethylene glycol. In a castrated rat model the Spiegelmer further demonstrated strong GnRH antagonist activity, which is more pronounced and persists longer with the polyethylene glycol-linked derivative. Furthermore, in rabbits the anti-GnRH Spiegelmer was shown to have a very low, possibly negligible immunogenic potential. These studies suggest that Spiegelmers could be of substantial interest in the development of new pharmaceutical approaches against GnRH and other targets.

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.

Immune and Inflammatory Cell Involvement in the Pathology of Idiopathic Pulmonary Arterial Hypertension

RATIONALE Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and vascular remodeling. Recent studies have revealed that immune and inflammatory responses play a crucial role in pathogenesis of idiopathic PAH. OBJECTIVES To systematically evaluate the number and cross-sectional distribution of inflammatory cells in different sizes of pulmonary arteries from explanted lungs of patients with idiopathic PAH versus healthy donor lungs and to demonstrate functional relevance by blocking stromal-derived factor-1 by the Spiegelmer NOX-A12 in monocrotaline-induced pulmonary hypertension in rats. METHODS Immunohistochemistry was performed on lung tissue sections from patients with idiopathic PAH and healthy donors. All positively stained cells in whole-lung tissue sections, surrounding the vessels, and in the different compartments of the vessels were counted. To study the effects of blocking SDF-1, rats with monocrotaline-induced pulmonary hypertension were treated with NOX-A12 from Day 21 to Day 35 after monocrotaline administration. MEASUREMENTS AND MAIN RESULTS We found a significant increase of the perivascular number of macrophages (CD68(+)), macrophages/monocytes (CD14(+)), mast cells (toluidine blue(+)), dendritic cells (CD209(+)), T cells (CD3(+)), cytotoxic T cells (CD8(+)), and helper T cells (CD4(+)) in vessels of idiopathic PAH lungs compared with control subjects. FoxP3(+) mononuclear cells were significantly decreased. In the monocrotaline model, the NOX-A12-induced reduction of mast cells, CD68(+) macrophages, and CD3(+) T cells was associated with improvement of hemodynamics and pulmonary vascular remodeling. CONCLUSIONS Our findings reveal altered perivascular inflammatory cell infiltration in pulmonary vascular lesions of patients with idiopathic pulmonary arterial hypertension. Targeting attraction of inflammatory cells by blocking stromal-derived factor-1 may be a novel approach for treatment of PAH.

Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist

We have developed an automated SELEX (Systematic Evolution of Ligands by EXponential Enrichment) process that allows the execution of in vitro selection cycles without any direct manual intervention steps. The automated selection protocol is designed to provide for high flexibility and versatility in terms of choice of buffers and reagents as well as stringency of selection conditions. Employing the automated SELEX process, we have identified RNA aptamers to the mirror-image configuration (d-peptide) of substance P. The peptide substance P belongs to the tachykinin family and exerts various biologically important functions, such as peripheral vasodilation, smooth muscle contraction and pain transmission. The aptamer that was identified most frequently was truncated to the 44mer SUP-A-004. The mirror-image configuration of SUP-A-004, the so-called Spiegelmer, has been shown to bind to naturally occurring l-substance P displaying a Kd of 40 nM and to inhibit (IC50 of 45 nM) l-substance P-mediated Ca2+ release in a cell culture assay.

GnRH binding RNA and DNA Spiegelmers: a novel approach toward GnRH antagonism.

Mirror-image oligonucleotide ligands (Spiegelmers) that bind to the pharmacologically relevant target gonadotropin-releasing hormone I (GnRH) with high affinity and high specificity have been identified using the Spiegelmer technology. GnRH is a decapeptide that plays an important role in mammalian reproduction and sexual maturation and is associated with several benign and malignant diseases. First, aptamers that bind to D-GnRH with dissociation constants of 50-100 nM were isolated out of RNA and DNA libraries. The respective enantiomers of the DNA and RNA aptamers were synthesized, and their binding to L-GnRH was shown. These Spiegelmers bind to L-GnRH with similar affinity to that of the corresponding aptamers that bind to D-GnRH. We further demonstrated dose-dependent inhibition of GnRH-induced Ca(2+) release in Chinese hamster ovary cells that were stably transfected with the human GnRH receptor.

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.

Dual Blockade of the Homeostatic Chemokine CXCL12 and the Proinflammatory Chemokine CCL2 Has Additive Protective Effects on Diabetic Kidney Disease

Monocyte/ chemoattractant protein-1/chemokine ligand (CCL) 2 and stromal cell-derived factor-1/CXCL12 both contribute to glomerulosclerosis in mice with type 2 diabetes mellitus, through different mechanisms. CCL2 mediates macrophage-related inflammation, whereas CXCL12 contributes to podocyte loss. Therefore, we hypothesized that dual antagonism of these chemokines might have additive protective effects on the progression of diabetic nephropathy. We used chemokine antagonists based on structured l-enantiomeric RNA (so-called Spiegelmers) ie, the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12. Male db/db mice, uninephrectomized at the age of 6 weeks, received injections of Spiegelmer, both Spiegelmers, nonfunctional control Spiegelmer, or vehicle from the age of 4 months for 8 weeks. Dual blockade was significantly more effective than monotherapy in preventing glomerulosclerosis. CCL2 blockade reduced glomerular leukocyte counts and renal-inducible nitric oxide synthase or IL-6 mRNA expression. CXCL12 blockade maintained podocyte numbers and renal nephrin and podocin mRNA expression. Consistently, CXCL12 blockade suppressed nephrin mRNA up-regulation in primary cultures of human glomerular progenitors induced to differentiate toward the podocyte lineage. All previously mentioned parameters were significantly improved in the dual-blockade group, which also suppressed proteinuria and was associated with the highest levels of glomerular filtration rate. Blood glucose levels and body weight were identical in all treatment groups. Dual chemokine blockade can have additive effects on the progression of diabetic kidney disease when the respective chemokine targets mediate different pathomechanisms of disease (ie, inflammation and progenitor differentiation toward the podocyte lineage).

Turning mirror-image oligonucleotides into drugs: the evolution of Spiegelmer® therapeutics ☆

Spiegelmers are synthetic target-binding oligonucleotides built from non-natural l-nucleotides. Like aptamers, Spiegelmers fold into distinct shapes that bind the targets with high affinity and selectivity. Furthermore, the mirror-image configuration confers plasma stability and immunological passivity. Various Spiegelmers against pharmacologically attractive targets were shown to be efficacious in animal models. Three Spiegelmer candidates: emapticap pegol (NOX-E36; anti-CCL2), olaptesed pegol (NOX-A12; anti-CXCL12) and lexaptepid pegol (NOX-H94; anti-hepcidin), underwent regulatory safety studies, demonstrated good safety profiles in healthy volunteers and were taken into Phase IIa studies in patients. Proof-of-concept for emapticap pegol has recently been demonstrated in diabetic nephropathy patients. Furthermore, promising interim Phase IIa data of olaptesed pegol and lexapteptid pegol also suggest efficacy in the respective patient populations.

Neonatal ghrelin programs development of hypothalamic feeding circuits

A complex neural network regulates body weight and energy balance, and dysfunction in the communication between the gut and this neural network is associated with metabolic diseases, such as obesity. The stomach-derived hormone ghrelin stimulates appetite through interactions with neurons in the arcuate nucleus of the hypothalamus (ARH). Here, we evaluated the physiological and neurobiological contribution of ghrelin during development by specifically blocking ghrelin action during early postnatal development in mice. Ghrelin blockade in neonatal mice resulted in enhanced ARH neural projections and long-term metabolic effects, including increased body weight, visceral fat, and blood glucose levels and decreased leptin sensitivity. In addition, chronic administration of ghrelin during postnatal life impaired the normal development of ARH projections and caused metabolic dysfunction. Consistent with these observations, direct exposure of postnatal ARH neuronal explants to ghrelin blunted axonal growth and blocked the neurotrophic effect of the adipocyte-derived hormone leptin. Moreover, chronic ghrelin exposure in neonatal mice also attenuated leptin-induced STAT3 signaling in ARH neurons. Collectively, these data reveal that ghrelin plays an inhibitory role in the development of hypothalamic neural circuits and suggest that proper expression of ghrelin during neonatal life is pivotal for lifelong metabolic regulation.