The role of antidrug antibodies in ustekinumab therapy and the impact of methotrexate.

OBJECTIVE: We investigated the impact of concomitant MTX on ustekinumab (UST) levels and antidrug antibody (ADA) formation in PsA and evaluated consequences in pharmacodynamics and pharmacokinetics. METHODS: We conducted a post-hoc analysis on 112 PsA serum samples of subjects treated with open-label UST and either concomitant MTX (UST/MTX, n = 58) or placebo (UST/pbo, n = 54) obtained in a randomized (1:1), double-blind, multicentre trial. A validated antibody-binding-based multitiered testing was used to detect ADA and ADA with neutralizing capacity (nADA). The impact of MTX on UST immunogenicity was analysed by comparison of UST/pbo with UST/MTX cohorts at different time points. Patient- and disease-related predispositions for ADA formation were investigated with multiple linear regression analysis. Immunogenicity impact on pharmacokinetics, safety and efficacy was determined by cohort comparison between patients with and without ADA formation. RESULTS: Over 52 weeks, 11 UST/pbo- and 19 UST/MTX-treated patients developed ADA (P > 0.05). In the UST/pbo cohort, the visit-dependent UST levels were in the range of 0.047 (0.05) -0.110 (0.07) µg/ml overall, and 0.037 (0.04)-0.091 (0.08) µg/ml in ADA-confirmed subjects. In UST/MTX-treated patients, the UST levels exhibited an intervisit variation in the range of 0.0502 (0.04)-0.106 (0.07) µg/ml overall and 0.029 (0.03)-0.097 (0.07) µg/ml in ADA positive subjects (P > 0.05). At week 52, ADA-confirmed patients did not differ significantly (P > 0.05) in safety or clinical outcomes from ADA-negative patients. CONCLUSION: Concomitant MTX had no significant impact on UST immunogenicity. Furthermore, ADA formation was not associated with impairments in UST safety, efficacy or trough levels. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT03148860.

Comparing the Effects of Rocaglates on Energy Metabolism and Immune Modulation on Cells of the Human Immune System.

A promising new approach to broad spectrum antiviral drugs is the inhibition of the eukaryotic translation initiation factor 4A (elF4A), a DEAD-box RNA helicase that effectively reduces the replication of several pathogenic virus types. Beside the antipathogenic effect, modulation of a host enzyme activity could also have an impact on the immune system. Therefore, we performed a comprehensive study on the influence of elF4A inhibition with natural and synthetic rocaglates on various immune cells. The effect of the rocaglates zotatifin, silvestrol and CR-31-B (-), as well as the nonactive enantiomer CR-31-B (+), on the expression of surface markers, release of cytokines, proliferation, inflammatory mediators and metabolic activity in primary human monocyte-derived macrophages (MdMs), monocyte-derived dendritic cells (MdDCs), T cells and B cells was assessed. The inhibition of elF4A reduced the inflammatory potential and energy metabolism of M1 MdMs, whereas in M2 MdMs, drug-specific and less target-specific effects were observed. Rocaglate treatment also reduced the inflammatory potential of activated MdDCs by altering cytokine release. In T cells, the inhibition of elF4A impaired their activation by reducing the proliferation rate, expression of CD25 and cytokine release. The inhibition of elF4A further reduced B-cell proliferation, plasma cell formation and the release of immune globulins. In conclusion, the inhibition of the elF4A RNA helicase with rocaglates suppressed the function of M1 MdMs, MdDCs, T cells and B cells. This suggests that rocaglates, while inhibiting viral replication, may also suppress bystander tissue injury by the host immune system. Thus, dosing of rocaglates would need to be adjusted to prevent excessive immune suppression without reducing their antiviral activity.

The Pharmacological Potential of Novel Melittin Variants from the Honeybee and Solitary Bees against Inflammation and Cancer.

The venom of honeybees is composed of numerous peptides and proteins and has been used for decades as an anti-inflammatory and anti-cancer agent in traditional medicine. However, the bioactivity of specific biomolecular components has been evaluated for the predominant constituent, melittin. So far, only a few melittin-like peptides from solitary bee species have been investigated, and the molecular mechanisms of bee venoms as therapeutic agents remain largely unknown. Here, the preclinical pharmacological activities of known and proteo-transcriptomically discovered new melittin variants from the honeybee and more ancestral variants from phylogenetically older solitary bees were explored in the context of cancer and inflammation. We studied the effects of melittin peptides on cytotoxicity, second messenger release, and inflammatory markers using primary human cells, non-cancer, and cancerous cell lines. Melittin and some of its variants showed cytotoxic effects, induced Ca2+ signaling and inhibited cAMP production, and prevented LPS-induced NO synthesis but did not affect the IP3 signaling and pro-inflammatory activation of endothelial cells. Compared to the originally-described melittin, some phylogenetically more ancestral variants from solitary bees offer potential therapeutic modalities in modulating the in vitro inflammatory processes, and hindering cancer cell viability/proliferation, including aggressive breast cancers, and are worth further investigation.

In Vitro Safety, Off-Target and Bioavailability Profile of the Antiviral Compound Silvestrol.

We characterized the in vitro safety and bioavailability profile of silvestrol, a compound effective against various viruses, such as corona- and Ebolaviruses, with an EC50 value of about 5 nM. The cytotoxic profile of silvestrol was assessed in various cancer cell lines, as well as the mutagenic and genotoxic potential with Ames and micronuclei tests, respectively. To identify off-target effects, we investigated whether silvestrol modulates G-protein coupled receptor (GPCR) signaling pathways. To predict the bioavailability of silvestrol, its stability, permeability and cellular uptake were determined. Silvestrol reduced viability in a cell-type-dependent manner, mediated no off-target effects via GPCRs, had no mutagenic potential and minor genotoxic effects at 50 nM. Silvestrol did not disturb cell barrier integrity, showed low membrane permeability, was stable in liver microsomes and exhibited good cellular uptake. Efficient cellular uptake and increased cytotoxicity were observed in cell lines with a low expression level of the transport protein P-glycoprotein, the known efflux transporter of silvestrol. In conclusion, silvestrol showed low permeability but good cellular uptake and high stability. Cell-type-dependent cytotoxicity seems to be caused by the accumulation of silvestrol in cells lacking the ability to expel silvestrol due to low P-glycoprotein levels.

Lecanoric acid mediates anti-proliferative effects by an M phase arrest in colon cancer cells.

Lichen extracts containing, among other compounds, depsides such as evernic acid, atranorin, and lecanoric acid possess anti-proliferative effects. We aimed to identify lichen metabolites that are responsible for the observed anti-proliferative effects. We performed cytotoxicity, cell colony, cell cycle and apoptosis assays in various cell lines or primary immune cells. We analyzed several cell cycle proteins and apoptosis-related proteins to gain insights into the underlying mechanism. All depsides reduced the viability of the tested cell lines (HCT-116, HEK293T, HeLa, NIH3T3, RAW246.7) in a cell line-dependent manner with lecanoric acid being the most effective. Atranorin did not influence the cell cycle or colony formation in HCT-116 cells, but induced apoptosis in HCT-116 cells. Evernic acid showed no anti-proliferative effects. Lecanoric acid inhibited cell colony formation already at 0.03 µg/ml in HCT-116 cells and induced a G2 cell cycle block in several cell lines. Moreover, lecanoric acid arrested the cell cycle, presumably in the M phase, since expression of cyclin B1 and phosphorylated histone H3 was upregulated, whereas the inactive cyclin-dependent kinase 1 (CDK1) was reduced in HCT-116 cells. Most importantly, cell death induced by lecanoric acid was more prominent in cancer cells than in primary human immune and endothelial cells. In conclusion, lecanoric acid seems to mediate its anti-proliferative effects via arrest of cells in the M phase. Our data suggest lecanoric acid may be a potential new candidate for anti-cancer therapy, because it has anti-proliferative effects on cancer cell lines, and does not affect primary immune cells.

T-Cell-Specific CerS4 Depletion Prolonged Inflammation and Enhanced Tumor Burden in the AOM/DSS-Induced CAC Model.

To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.

Cytoreductive treatment in real life: a chart review analysis on 1440 patients with polycythemia vera.

PURPOSE: Patients with polycythemia vera (PV) show an elevated incidence of thromboembolic complications and decreased survival when compared to age-matched healthy individuals. Hypercellularity as indicated by elevated hematocrit, pathophysiological changes induced by the JAK2 driver mutation and cardiovascular risk factors contribute to the increased incidence of thromboembolic events. Higher age and a history of thromboembolic events define a high-risk population of PV patients. Depending on the individual risk profile, phlebotomy or pharmacologic cytoreduction is recommended in combination with low-dose acetylsalicylic acid. Stringent cytoreduction is required for effective risk reduction. However, in recent reports, the rate of thromboembolic complications in PV patients under cytoreductive therapy appears still elevated compared to healthy individuals. This study reports on a chart review to assess for cytoreductive therapy of 1440 PV patients in real life. METHODS: Forty-two eligible hematologists/oncologists in private practice treating patients with MPN were recruited to participate in a paper-pencil-based survey conducted between January 2019 and March 2020 in Germany. Physicians were asked to report primary documented data obtained from patient charts. Descriptive analyses were conducted to assess for patient characteristics, treatment modalities, risk factors and thromboembolic complications. RESULTS: Data were collected from the patient charts of 1440 individuals diagnosed with PV. The patient population was older than those reported in multicenter trials with a median age of 72.2 years at the time of reporting and 63.5 years at diagnosis. Age was the main factor accounting for high-risk status with 84.7% of patients being above the age of 60 followed by thromboembolic complications reported in 21.3% of patients. The use of pharmacologic cytoreduction was highly variable between participating centers with an average of 60.7% and a range of 10.1-100%. Hydroxyurea was the most frequently used drug followed by ruxolitinib, while interferons were reported for a minority of patients. For 35.4% of patients a persistent need for phlebotomy in addition to cytoreductive treatment was reported. Although presence of high-risk criteria and insufficient disease control were reported as main triggers to initiate pharmacologic cytoreduction, 28.1% had elevated hematocrit values (> 45%) and 38.6% showed persistence of elevated leukocyte count (> 109/l) while on cytoreductive treatment. In contrast, physician-reported symptom burden was lower than published in clinical trials and patient-reported outcomes. The rate of patients experiencing thromboembolic complications was 32.2% at any time and 14.3% after diagnosis with most patients receiving acetylsalicylic acid and 10.8% remaining on oral anticoagulants or heparin. CONCLUSIONS: Cytoreductive treatment of high-risk PV in real life is highly variable regarding indication for cytoreduction and definition of therapy resistance. This study highlights the need for (i) improved risk stratification for thromboembolic events, (ii) consequent indication of pharmacologic cytoreduction in high-risk PV and (iii) attention to signs of therapy resistance that can trigger an earlier and stringent switch to second line agents.

Characterization of ACE Inhibitors and AT1R Antagonists with Regard to Their Effect on ACE2 Expression and Infection with SARS-CoV-2 Using a Caco-2 Cell Model.

Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin-renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT1 receptor (AT1R). METHODS: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model. RESULTS: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate. CONCLUSION: ACE inhibitors and AT1R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.

Sodium Bituminosulfonate Used to Treat Rosacea Modulates Generation of Inflammatory Mediators by Primary Human Neutrophils.

BACKGROUND: Sodium bituminosulfonate is derived from naturally occurring sulphur-rich oil shale and is used for the treatment of the inflammatory skin disease rosacea. Major molecular players in the development of rosacea include the release of enzymes that process antimicrobial peptides which, together with reactive oxygen species (ROS) and vascular endothelial growth factor (VEGF), promote pro-inflammatory processes and angiogenesis. The aim of this study was to address the molecular mechanism(s) underlying the therapeutic benefit of the formulation sodium bituminosulfonate dry substance (SBDS), which is indicated for the treatment of skin inflammation, including rosacea. METHODS: We investigated whether SBDS regulates the expression of cytokines, the release of the antimicrobial peptide LL-37, calcium mobilization, proteases (matrix metalloproteinase, elastase, kallikrein (KLK)5), VEGF or ROS in primary human neutrophils. In addition, activity assays with 5-lipoxygenase (5-LO) and recombinant human MMP9 and KLK5 were performed. RESULTS: We observed that SBDS reduces the release of the antimicrobial peptide LL-37, calcium, elastase, ROS and VEGF from neutrophils. Moreover, KLK5, the enzyme that converts cathelicidin to LL-37, and 5-LO that produces leukotriene (LT)A4, the precursor of LTB4, were both inhibited by SBDS with an IC50 of 7.6 µg/mL and 33 µg/mL, respectively. CONCLUSION: Since LTB4 induces LL-37 which, in turn, promotes increased intracellular calcium levels and thereby, ROS/VEGF/elastase release, SBDS possibly regulates the LTB4/LL-37/calcium - ROS/VEGF/elastase axis by inhibiting 5-LO and KLK5. Additional direct effects on other pro-inflammatory pathways such as ROS generation cannot be ruled out. In summary, SBDS reduces the generation of inflammatory mediators from human neutrophils possibly accounting for its anti-inflammatory effects in rosacea.

The immunomodulatory potential of the arylmethylaminosteroid sc1o.

Developing resistance mechanisms of pathogens against established and frequently used drugs are a growing global health problem. Besides the development of novel drug candidates per se, new approaches to counteract resistance mechanisms are needed. Drug candidates that not only target the pathogens directly but also modify the host immune system might boost anti-parasitic defence and facilitate clearance of pathogens. In this study, we investigated whether the novel anti-parasitic steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni, might exhibit immunomodulatory properties. Our results reveal that 50 µM sc1o amplified the inflammatory potential of M1 macrophages and shifted M2 macrophages in a pro-inflammatory direction. Since M1 macrophages used predominantly glycolysis as an energy source, it is noteworthy that sc1o increased glycolysis and decreased oxidative phosphorylation in M2 macrophages. The effect of sc1o on the differentiation and activation of dendritic cells was ambiguous, since both pro- and anti-inflammatory markers were regulated. In conclusion, sc1o has several immunomodulatory effects that could possibly assist the immune system by counteracting the anti-inflammatory immune escape strategy of the parasite P. falciparum or by increasing pro-inflammatory mechanisms against pathogens, albeit at a higher concentration than that required for the anti-parasitic effect. KEY MESSAGES: • The anti-parasitic steroid compound 1o (sc1o) can modulate human immune cells. • Sc1o amplified the potential of M1 macrophages. • Sc1o shifts M2 macrophages to a M1 phenotype. • Dendritic cell differentiation and activation was ambiguously modulated. • Administration of sc1o could possibly assist the anti-parasitic defence.

Unraveling the Pharmacological Potential of Lichen Extracts in the Context of Cancer and Inflammation With a Broad Screening Approach.

Lichen-forming fungi are symbiotic organisms that synthesize unique natural products with potential for new drug leads. Here, we explored the pharmacological activity of six lichen extracts (Evernia prunastri, Pseudevernia furfuracea, Umbilicaria pustulata, Umbilicaria crustulosa, Flavoparmelia caperata, Platismatia glauca) in the context of cancer and inflammation using a comprehensive set of 11 functional and biochemical in vitro screening assays. We assayed intracellular Ca2+ levels and cell migration. For cancer, we measured tumor cell proliferation, cell cycle distribution and apoptosis, as well as the angiogenesis-associated proliferation of endothelial cells (ECs). Targeting inflammation, we assayed leukocyte adhesion onto ECs, EC adhesion molecule expression, as well as nitric oxide production and prostaglandin (PG)E2 synthesis in leukocytes. Remarkably, none of the lichen extracts showed any detrimental influence on the viability of ECs. We showed for the first time that extracts of F. caperata induce Ca2+ signaling. Furthermore, extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca reduced cell migration. Interestingly, F. caperata extracts strongly decreased tumor cell survival. The proliferation of ECs was significantly reduced by E. prunastri, P. furfuracea, and F. caperata extracts. The extracts did not inhibit the activity of inflammatory processes in ECs. However, the pro-inflammatory activation of leukocytes was inhibited by extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca. After revealing the potential biological activities of lichen extracts by an array of screening tests, a correlation analysis was performed to evaluate particular roles of abundant lichen secondary metabolites, such as atranorin, physodic acid, and protocetraric acid as well as usnic acid in various combinations. Overall, some of the lichen extracts tested in this study exhibit significant pharmacological activity in the context of inflammation and/or cancer, indicating that the group lichen-forming fungi includes promising members for further testing.

Ceramide Synthase 5 Deficiency Aggravates Dextran Sodium Sulfate-Induced Colitis and Colon Carcinogenesis and Impairs T-Cell Activation.

Ceramide synthase 5 is one of six enzymes that catalyze the production of ceramides from sphingosine or sphinganine. Ceramides are important components of cell membranes and act as signaling molecules. Previously it has been shown that ceramide synthase 6 and 2 influence colitis in several animal models with sometimes opposite effects. Here, we investigated the disease course of dextran sodium sulfate-induced acute colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer in mice with global ceramide synthase 5 knockout (CerS5-ko) or with ceramide synthase 5 knockout restricted to the colon epithelium (CerS5fl/fl VilCre). We monitored disease development and analyzed colon barrier function as well as the immune cell status in these mice. CerS5-ko mice but not CerS5fl/fl-VilCre mice were more susceptible to acute and chronic inflammation. However, the cell barrier function of colon epithelial cells was not disturbed by downregulation of ceramide synthase 5. Instead, untreated CerS5-ko mice displayed reduced numbers of CD3+ immune cells in the spleen, colon, and blood, especially of intraepithelial CD8+ T-cells, which was not obvious in CerS5fl/fl Vil Cre mice. Reduced T-cell number in colon tissue of CerS5-ko mice was accompanied by a reduced expression of IL-1ß, IFNγ, and IL-4. In vitro investigations revealed that knockdown of ceramide synthase 5 in T-cells impaired T-cell activation. In summary, we show that CerS5-ko mice were more susceptible to dextran sodium sulfate-induced colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer. A reduced number of T-cells in the colon epithelium that was already the case in untreated CerS5-ko mice might have contributed to this effect.

In-vitro safety and off-target profile of the anti-parasitic arylmethylaminosteroid 1o.

Parasite-mediated diseases like malaria and schistosomiasis are growing health problems worldwide and novel drug candidates are urgently needed. In this study, the in-vitro safety profile of steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni with an IC50 value of 5 nM, was characterized. We assessed viability/proliferation, apoptosis and cell cycle tests to determine the cytotoxic profile of sc1o in cancer cells. The mutagenic potential was determined with the AMES test. To identify off-target effects we investigated whether sc1o interacts with safety-relevant molecules such as cytochrome P450 (CYP) enzymes, phosphodiesterases (PDE), histone deacteylases (HDAC) and human ether-a-go-go related gene (hERG). Furthermore, to predict the potential bioavailability of sc1o, its effect on Caco-2 cell barrier integrity, by measurement of the transepithelial electrical resistance (TEER), was determined. Sc1o at 25 µM reduced cell viability, probably through cell-cycle arrest, but did not induce apoptosis in cancer cells. No adverse off-target effects nor mutagenic potential of sc1o were observed. Furthermore, sc1o did not disturb the integrity of the cell barrier, but exhibited low membrane permeability, apparently due to cell adherence. In conclusion, sc1o up to 10 µM showed a good in-vitro safety profile.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Role of ceramide synthase 2 in G-CSF signaling and G-CSF-R translocation into detergent-resistant membranes.

Ceramides are sphingolipids with defined acyl chain lengths, which are produced by corresponding ceramide synthases (CerS1-6). In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), the ablation of CerS2 suppresses EAE-pathology by reducing neutrophil migration into the central nervous system. This migration is induced by granulocyte-colony stimulating factor (G-CSF) signaling. G-CSF signaling leads to a signal cascade including the phosphorylation of Lyn kinase and STAT3. This in turn regulates expression of the neutrophil surface receptor chemokine receptor 2 (CXCR2) and causes translocation of the receptor into detergent-resistant membranes (DRMs). In this study we investigated the role of ceramides in G-CSF signaling. We found, that G-CSF treatment of wild type bone marrow cells (BMCs) leads to translocation of G-CSF-receptor (G-CSF-R) into DRMs. G-CSF also induces downregulation of ceramides in WT and CerS2 null BMCs, as well as upregulation of very long chain lactosylceramides. However, in CerS2 null BMCs, G-CSF failed to induce translocation of G-CSF-R into DRMs, leading to reduced phosphorylation of Lyn and reduced CXCR2 expression. Interestingly, G-CSF signaling in CerS6 null BMCs was not affected. In conclusion, very long chain ceramides are important for G-CSF signaling and translocation of G-CSF-R into DRMs.

Stimulating brain recovery after stroke using theranostic albumin nanocarriers loaded with nerve growth factor in combination therapy.

For many years, delivering drug molecules across the blood brain barrier has been a major challenge. The neuropeptide nerve growth factor is involved in the regulation of growth and differentiation of cholinergic neurons and holds great potential in the treatment of stroke. However, as with many other compounds, the biomolecule is not able to enter the central nervous system. In the present study, nerve growth factor and ultra-small particles of iron oxide were co-encapsulated into a chemically crosslinked albumin nanocarrier matrix which was modified on the surface with apolipoprotein E. These biodegradable nanoparticles with a size of 212 ±â€¯1 nm exhibited monodisperse size distribution and low toxicity. They delivered NGF through an artificial blood brain barrier and were able to induce neurite outgrowth in PC12 cells in vitro. In an animal model of stroke, the infarct size was significantly reduced compared to the vehicle control. The combination therapy of NGF and the small-molecular MEK inhibitor U0126 showed a slight but not significant difference compared to U0126 alone. However, further in vivo evidence suggests that successful delivery of the neuropeptide is possible as well as the synergism between those two treatments.

The relevance of ceramides and their synthesizing enzymes for multiple sclerosis.

Ceramide synthases (CerS) synthesize chain length specific ceramides (Cer), which mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that the genetic deletion of CerS2 suppresses EAE pathology by interaction with granulocyte-colony stimulating factor (G-CSF) signaling and CXC motif chemokine receptor 2 (CXCR2) expression, leading to impaired neutrophil migration. In the present study, we investigated the importance of Cers and their synthesizing/metabolizing enzymes in MS. For this purpose, a longitudinal study with 72 MS patients and 25 healthy volunteers was performed. Blood samples were collected from healthy controls and MS patients over 1- or 3-year periods, respectively. Immune cells were counted using flow cytometry, ceramide levels were determined using liquid chromatography-tandem mass spectrometry, and mRNA expression was analyzed using quantitative PCR. In white blood cells, C16-LacCer and C24-Cer were down-regulated in MS patients in comparison with healthy controls. In plasma, C16-Cer, C24:1-Cer, C16-GluCer, and C24:1-GluCer were up-regulated and C16-LacCer was down-regulated in MS patients in comparison with healthy controls. Blood samples from MS patients were characterized by an increased B-cell number. However, there was no correlation between B-cell number and Cer levels. mRNA expression of Cer metabolizing enzymes and G-CSF signaling enzymes was significantly increased in MS patients. Interestingly, G-CSF receptor (G-CSFR) and CXCR2 mRNA expression correlated with CerS2 and UDP-glucose Cer glucosyltransferase (UGCG) mRNA expression. In conclusion, our results indicate that Cer metabolism is linked to G-CSF signaling in MS.

Alpha-methylacyl-CoA racemase deletion has mutually counteracting effects on T-cell responses, associated with unchanged course of EAE.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Altering the metabolism of immune cells is an attractive strategy to modify their activity during autoimmunity in MS. We investigated the effect of modulating fatty acid metabolism in an animal model of MS, EAE. Alpha-methylacyl-CoA racemase (AMACR) converts R-configuration branched fatty acids into the S-configuration, thereby preparing them for ß-oxidation. We observed a significant, disease-dependent elevation of AMACR expression in monocytes and T cells from blood, draining lymph nodes and spleen of EAE mice during the preclinical phase. In vitro analysis revealed that the proliferation of T cells was inhibited in AMACR KO mice, but T-cell polarization was switched toward a pathogenic state involving the production of more IFN-γ and IL-17, but less IL-4. These opposing effects appeared to cancel out each other in vivo, because AMACR KO EAE mice showed a marginal increase in the severity of early clinical symptoms. AMACR was not regulated in the white blood cells of MS patients. Our data show that AMACR is regulated in immune cells during EAE, but it is not a suitable target for the treatment of MS due to its opposing effects.

Drug Release and Targeting: the Versatility of Polymethacrylate Nanoparticles for Peroral Administration Revealed by Using an Optimized In Vitro-Toolbox.

PURPOSE: The contribution of permeability and drug release to drug targeting were investigated in the course of development of a nanosized formulation of the anti-inflammatory compound TMP-001, for the local treatment in the gastrointestinal tract. METHODS: TMP-001 was encapsulated by nanoprecipitation into Eudragit® RS 100. The permeability of these carriers was investigated in an Ussing chamber model and the release rate was determined under biorelevant conditions. Formulation toxicity and particle-cell-interaction were investigated by flow cytometry, fluorescence and electron microscopy. Furthermore, spray drying was performed. RESULTS: Effective internalization of Eudragit®-nanoparticles into cancer cells was demonstrated. A burst release of the nanoparticles implied poor interaction of TMP-001 with Eudragit®. A sustained release (70.5% release after 30 min compared to 98.0% for the API) was accomplished after spray drying yielded an increased particle size. Recovery rate of TMP-001 after spray drying was 94.2 ± 5.9%. CONCLUSION: The release of API from polymeric nanoparticles contributes profoundly to the in vivo-performance of drug delivery devices in the gastrointestinal tract. The impact of drug-polymer interaction and particle size was analyzed. Sustained release of TMP-001 could only be achieved by increasing particle size. Therefore, biorelevant release testing has been demonstrated to be a valid tool for nanoformulation design.

Exacerbation of experimental autoimmune encephalomyelitis in ceramide synthase 6 knockout mice is associated with enhanced activation/migration of neutrophils.

Ceramides are mediators of inflammatory processes. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed that CerS6 mRNA expression was upregulated 15-fold in peripheral blood leukocytes before the onset of EAE symptoms. In peripheral blood leukocytes from MS patients, a 3.9-fold upregulation was found. Total genetic deletion of CerS6 and the selective deletion of CerS6 in peripheral blood leucocytes exacerbated the progression of clinical symptoms in EAE mice. This was associated with enhanced leukocyte, predominantly neutrophil infiltration and enhanced demyelination in the lumbar spinal cord of EAE mice. Interferon-gamma/tumor necrosis factor alpha (IFN-γ/TNF-α) and granulocyte colony-stimulating factor (G-CSF) both drive EAE development and induce expression of the integrin CD11b and the chemokine receptor C-X-C motif chemokine receptor 2 (CXCR2), and we found they also induce CerS6 expression. In vivo, the genetic deletion of CerS6 enhanced the activation/migration of neutrophils, as reflected by an enhanced upregulation of CD11b and CXCR2. In vitro, the genetic deletion of CerS6 enhanced the activation status of IFN-γ/TNF-α-stimulated neutrophils, as shown by increased expression of nitric oxide and CD11b and an increased adhesion capacity. In G-CSF-stimulated neutrophils, the migration status was enhanced, as reflected by an elevated level of CXCR2 and an increased migration capacity. These data suggest that CerS6/C16-Cer mediates feedback regulation by inhibiting the formation of CD11b and CXCR2, which are induced either by IFN-γ/TNF-α or by G-CSF, respectively. We conclude that CerS6/C16-Cer mediates anti-inflammatory effects during the development of EAE and MS possibly by suppressing the migration and deactivation of neutrophils.