Christiane Mühle

Functional Inhibitors of Acid Sphingomyelinase (FIASMAs): a novel pharmacological group of drugs with broad clinical applications.

Acid sphingomyelinase (ASM) is an important lipid-metabolizing enzyme cleaving sphingomyelin to ceramide, mainly within lysosomes. Acid ceramidase (AC) further degrades ceramide to sphingosine which can then be phosphorylated to sphingosine-1-phosphate. Ceramide and its metabolite sphingosine-1-phosphate have been shown to antagonistically regulate apoptosis, cellular differentiation, proliferation and cell migration. Inhibitors of ASM or AC therefore hold promise for a number of new clinical therapies, e.g. for Alzheimer’s disease and major depression on the one hand and cancer on the other. Inhibitors of ASM have been known for a long time. Cationic amphiphilic substances induce the detachment of ASM protein from inner lysosomal membranes with its consecutive inactivation, thereby working as functional inhibitors of ASM. We recently experimentally identified a large number of hitherto unknown functional inhibitors of ASM and determined specific physicochemical properties of such cationic amphiphilic substances that functionally inhibit ASM. We propose the acronym “FIASMA” (Functional Inhibitor of Acid SphingoMyelinAse) for members of this large group of compounds with a broad range of new clinical indications. FIASMAs differ markedly with respect to molecular structure and current clinical indication. Most of the available FIASMAs are licensed for medical use in humans, are minimally toxic and may therefore be applied for disease states associated with increased activity of ASM.

Brain membrane lipids in major depression and anxiety disorders

Major depression and anxiety disorders have high prevalence rates and are frequently comorbid. The neurobiological bases for these disorders are not fully understood, and available treatments are not always effective. Current models assume that dysfunctions in neuronal proteins and peptide activities are the primary causes of these disorders. Brain lipids determine the localization and function of proteins in the cell membrane and in doing so regulate synaptic throughput in neurons. Lipids may also leave the membrane as transmitters and relay signals from the membrane to intracellular compartments or to other cells. Here we review how membrane lipids, which play roles in the membranes function as a barrier and a signaling medium for classical transmitter signaling, contribute to depression and anxiety disorders and how this role may provide targets for lipid-based treatment approaches. Preclinical findings have suggested a crucial role for the membrane-forming n-3 polyunsaturated fatty acids, glycerolipids, glycerophospholipids, and sphingolipids in the induction of depression- and anxiety-related behaviors. These polyunsaturated fatty acids also offer new treatment options such as targeted dietary supplementation or pharmacological interference with lipid-regulating enzymes. While clinical trials support this view, effective lipid-based therapies may need more individualized approaches. Altogether, accumulating evidence suggests a crucial role for membrane lipids in the pathogenesis of depression and anxiety disorders; these lipids could be exploited for improved prevention and treatment. This article is part of a Special Issue entitled Brain Lipids.

Identification of Novel Functional Inhibitors of Acid Sphingomyelinase

We describe a hitherto unknown feature for 27 small drug-like molecules, namely functional inhibition of acid sphingomyelinase (ASM). These entities named FIASMAs (Functional Inhibitors of Acid SphingoMyelinAse), therefore, can be potentially used to treat diseases associated with enhanced activity of ASM, such as Alzheimers disease, major depression, radiation- and chemotherapy-induced apoptosis and endotoxic shock syndrome. Residual activity of ASM measured in the presence of 10 µM drug concentration shows a bimodal distribution; thus the tested drugs can be classified into two groups with lower and higher inhibitory activity. All FIASMAs share distinct physicochemical properties in showing lipophilic and weakly basic properties. Hierarchical clustering of Tanimoto coefficients revealed that FIASMAs occur among drugs of various chemical scaffolds. Moreover, FIASMAs more frequently violate Lipinskis Rule-of-Five than compounds without effect on ASM. Inhibition of ASM appears to be associated with good permeability across the blood-brain barrier. In the present investigation, we developed a novel structure-property-activity relationship by using a random forest-based binary classification learner. Virtual screening revealed that only six out of 768 (0.78%) compounds of natural products functionally inhibit ASM, whereas this inhibitory activity occurs in 135 out of 2028 (6.66%) drugs licensed for medical use in humans.

Cell‐based resurfacing of large cartilage defects: Long‐term evaluation of grafts from autologous transgene‐activated periosteal cells in a porcine model of osteoarthritis

OBJECTIVE To investigate the potential of transgene-activated periosteal cells for permanently resurfacing large partial-thickness cartilage defects. METHODS In miniature pigs, autologous periosteal cells stimulated ex vivo by bone morphogenetic protein 2 gene transfer, using liposomes or a combination of adeno-associated virus (AAV) and adenovirus (Ad) vectors, were applied on a bioresorbable scaffold to chondral lesions comprising the entire medial half of the patella. The resulting repair tissue was assessed, 6 and 26 weeks after transplantation, by histochemical and immunohistochemical methods. The biomechanical properties of the repair tissue were characterized by nanoindentation measurements. Implants of unstimulated cells and untreated lesions served as controls. RESULTS All grafts showed satisfactory integration into the preexisting cartilage. Six weeks after transplantation, AAV/Ad-stimulated periosteal cells had adopted a chondrocyte-like phenotype in all layers; the newly formed matrix was rich in proteoglycans and type II collagen, and its contact stiffness was close to that of healthy hyaline cartilage. Unstimulated periosteal cells and cells activated by liposomal gene transfer formed only fibrocartilaginous repair tissue with minor contact stiffness. However, within 6 months following transplantation, the AAV/Ad-stimulated cells in the superficial zone tended to dedifferentiate, as indicated by a switch from type II to type I collagen synthesis and reduced contact stiffness. In deeper zones, these cells retained their chondrocytic phenotype, coinciding with positive staining for type II collagen in the matrix. CONCLUSION Large partial-thickness cartilage defects can be resurfaced efficiently with hyaline-like cartilage formed by transgene-activated periosteal cells. The long-term stability of the cartilage seems to depend on physicobiochemical factors that are active only in deeper zones of the cartilaginous tissue.

Sustained delivery of therapeutic concentrations of human clotting factor IX--a comparison of adenoviral and AAV vectors administered in utero.

Prenatal somatic gene therapy has been considered for genetic disorders presenting with morbidity at birth. Haemophilia is associated with an increased risk of catastrophic perinatal bleeding complications such as intracranial haemorrhage, which could be prevented by gene transfer in utero. Prenatal gene therapy may be more promising than postnatal treatment, as the fetus may be more amenable to uptake and integration of therapeutic DNA and the immaturity of its immune system may permit life‐long immune tolerance of the transgenic protein, thus avoiding the dominant problem in haemophilia treatment, the formation of inhibitory antibodies.

Secretory sphingomyelinase in health and disease

Abstract Acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, hydrolyzes sphingomyelin to ceramide and phosphorylcholine. In mammals, the expression of a single gene, SMPD1, results in two forms of the enzyme that differ in several characteristics. Lysosomal ASM (L-ASM) is located within the lysosome, requires no additional Zn2+ ions for activation and is glycosylated mainly with high-mannose oligosaccharides. By contrast, the secretory ASM (S-ASM) is located extracellularly, requires Zn2+ ions for activation, has a complex glycosylation pattern and has a longer in vivo half-life. In this review, we summarize current knowledge regarding the physiology and pathophysiology of S-ASM, including its sources and distribution, molecular and cellular mechanisms of generation and regulation and relevant in vitro and in vivo studies. Polymorphisms or mutations of SMPD1 lead to decreased S-ASM activity, as detected in patients with Niemann-Pick disease B. Thus, lower serum/plasma activities of S-ASM are trait markers. No genetic causes of increased S-ASM activity have been identified. Instead, elevated activity is the result of enhanced release (e.g., induced by lipopolysaccharide and cytokine stimulation) or increased enzyme activation (e.g., induced by oxidative stress). Increased S-ASM activity in serum or plasma is a state marker of a wide range of diseases. In particular, high S-ASM activity occurs in inflammation of the endothelium and liver. Several studies have demonstrated a correlation between S-ASM activity and mortality induced by severe inflammatory diseases. Serial measurements of S-ASM reveal prolonged activation and, therefore, the measurement of this enzyme may also provide information on past inflammatory processes. Thus, S-ASM may be both a promising clinical chemistry marker and a therapeutic target.

Serum acid sphingomyelinase is upregulated in chronic hepatitis C infection and non alcoholic fatty liver disease

UNLABELLED Sphingolipids constitute bioactive molecules with functional implications in homeostasis and pathogenesis of various diseases. However, the role of sphingolipids as possible disease biomarkers in chronic liver disease remains largely unexplored. In the present study we used mass spectrometry and spectrofluorometry methods in order to quantify various sphingolipid metabolites and also assess the activity of an important corresponding regulating enzyme in the serum of 72 healthy volunteers as compared to 69 patients with non-alcoholic fatty liver disease and 69 patients with chronic hepatitis C virus infection. Our results reveal a significant upregulation of acid sphingomyelinase in the serum of patients with chronic liver disease as compared to healthy individuals (p<0.001). Especially in chronic hepatitis C infection acid sphingomyelinase activity correlated significantly with markers of hepatic injury (r=0.312, p=0.009) and showed a high discriminative power. Accumulation of various (dihydro-) ceramide species was identified in the serum of patients with non-alcoholic fatty liver disease (p<0.001) and correlated significantly to cholesterol (r=0.448, p<0.001) but showed a significant accumulation in patients with normal cholesterol values as well (p<0.001). Sphingosine, a further bioactive metabolite, was also upregulated in chronic liver disease (p<0.001). However, no significant correlation to markers of hepatic injury was identified. CONCLUSION Chronic hepatitis C virus infection and non-alcoholic fatty liver disease induce a significant upregulation of serum acid sphingomyelinase which appears as a novel biomarker in chronic hepatopathies. Further studies are required to elucidate the potential of the sphingolipid signaling pathway as putative therapeutic target in chronic liver disease.

The role of ceramide in major depressive disorder

Major depression is a severe mood disorder with a lifetime prevalence of more than 10%. The pharmacokinetic hypothesis claims that a slow accumulation of antidepressant drugs by acid trapping mainly into lysosomes is responsible for the therapeutic latency and that a lysosomal target mediates the antidepressant effects. The lysosomal lipid metabolizing enzyme acid sphingomyelinase (ASM) cleaves sphingomyelin into ceramide and phosphorylcholine. In a pilot study, the activity of this enzyme was increased in peripheral blood cells of patients with major depressive disorder (MDD), making the ASM an interesting molecular target of antidepressant drugs. Indeed, several antidepressant drugs functionally inhibit ASM. The ASM/ceramide pathway might be a missing link unifying independent findings in neurobiology and the treatment of MDD such as therapeutic latency, oxidative stress, immune activation and increased risk of cardiovascular disease.

Activity of secretory sphingomyelinase is increased in plasma of alcohol-dependent patients.

BACKGROUND Acid sphingomyelinase (ASM, EC 3.1.4.12) hydrolyzes sphingomyelin to ceramide and represents a major regulator of sphingolipid metabolism. Increased activity of ASM has been observed in a variety of human diseases, and a critical contribution of ASM to medical conditions was demonstrated in several mouse models. In agreement with increased ASM activity in cell lines treated with ethanol, we have recently found higher levels of ASM activity in peripheral blood cells of active drinkers. However, the influence of ethanol on secretory ASM (S-ASM) has not been investigated so far. METHODS ASM activity and routine blood parameters were determined in plasma samples of 27 patients with alcohol dependence during physical withdrawal and compared to a group of 36 healthy volunteers. RESULTS Compared to the control group, patients with alcohol dependence had S-ASM activity increased by about 3-fold (141 ± 69 vs. 428 ± 220 pmol/ml/h; p < 0.001) at the beginning of physical withdrawal. During withdrawal, S-ASM activity decreased by about 50% (p < 0.001; day 0 vs. day 7 to 10) and finally approximated nearly normal values. On the day of admission, S-ASM activity correlated positively with levels of carbohydrate-deficient transferrin (r = 0.410, p = 0.034) and high-density lipoprotein cholesterol (r = 0.440, p = 0.022) and inversely with body mass index (r = -0.509; p = 0.007), glucose (r = -0.480; p = 0.011), triglycerides (r = -0.592; p = 0.001), and large unstained cells (r = -0.526; p = 0.017). CONCLUSIONS Activity of S-ASM is increased in alcohol-dependent patients and correlates with established biomarkers of excessive drinking. The increased S-ASM activity is implicated in alcohol-induced lipid alterations and might be relevant for the occurrence of alcohol-related disorders.

Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display.

Clotting factor VIII (fVIII)-inhibitory antibodies represent a major problem in the treatment of haemophilia A. To understand the inactivation mechanisms and to pave the way towards modifications of recombinant clotting factors that reduce their immunogenicity, the exact localization of immunodominant epitopes is required. Here, a random peptide phage display library was employed to identify epitopes of polyclonal fVIII antibodies isolated from patients plasma by affinity chromatography. FVIII-binding specificity and inhibitory activity of the isolated fVIII antibodies were confirmed by ELISA and Bethesda assays. Phage selection on the individual samples yielded several phages which were displaced from binding to the respective antibody preparation by fVIII. Their homology with amino acid motifs of human fVIII and immunoprecipitation results with radioactively labelled fVIII fragments suggested putative epitopes in the A1, A2 and C1 domains of fVIII for one and in the C2 domain for another patient. Synthetic peptides corresponding to the A2, C1 and C2 domain epitopes blocked antibody binding to fVIII and partially neutralized the inhibitory activity of the respective plasma in Bethesda assays. These results provide the proof of principle that random peptide libraries can be used for the mapping of epitopes in a polyclonal antibody preparation.