Novel Coupled Molecules from Active Structural Motifs of Synthetic and Natural Origin as Immunosuppressants

Introduction: Nitric oxide (NO) is an important mediator in the pathogenesis and control of immune system-related disorders and its levels are modulated by inducible NO synthase (iNOS). Oxidative stress is another pathological indication in majority of autoimmune disorders. The present study aims at the development of coupled molecules via selection of pharmacophores for both immunomodulatory and antioxidant activities through iNOS inhibition. Methods: Variedly substituted coumarin moieties are coupled with naturally occurring phenols through an amide linkage and were predicted for activities using computer-based program PASS. The compounds predicted to have dual activities were synthesized. Docking studies were carried out against iNOS (PDB 1R35) and compounds having good docking score were evaluated for immunomodulatory and antioxidant activities. Results: The synthesized compounds were found to be pure and were obtained in good yields. Compounds with maximum docking score (YR1a, YR2e, YR2c and YR4e) were selected for evaluation by in vitro models. Compounds YR2e and YR2c markedly inhibited the reduction of NBT dye and showed maximum % iNOS inhibition. In DPPH assay, compound YR4e was observed as the most potent antioxidant (EC50 0.33 µM/mL). Based on these studies, compounds YR2e and YR2c were selected for haemagglutination test. Compound YR2e was observed as the most active immunosuppressant with maximal inhibitory ability of iNOS and NBT reduction and lower HAT value of 3.5. Conclusion: Compound YR2e can be utilized as a pharmacological agent in the prevention or treatment of immunomodulatory diseases such as tumors, rheumatoid arthritis, ulcerative colitis, organ transplant and other autoimmune disorders.

The effect of acute somatic pain on the killing activity of neutrophils in newborn rats

The immune system is subject to all sorts of influences. Pain is one of them, accompanying an organism’s existence. It is essential to be aware of and account for age-related characteristics of the innate immunity in order to adequately assess their dynamics in ontogenesis. The literature is scarce on the changes to the killing activity of neutrophils occurring in newborns in response to acute pain. The aim of this study was to detect potential changes to the phagocytic activity of neutrophils in response to an algogenic stimulus in newborn rats. The experiments were carried out in 3-5-day-old rats. Two groups were formed: the control group and the main group, in which acute pain was modelled. Blood samples were collected 2, 30–60 and 120–180 minutes after exposure to the algogenic stimulus. The microbicidal activity of neutrophils was measured using a spectrophotometric modification of the spontaneous/stimulated nitroblue tetrazolium (NBT) reduction test. The results were compared using the Mann-Whitney U test. In the first hour following pain modeling, the stimulated NBT reduction test demonstrated an increase in the measured parameters from 71.5 to 87.4 a.u. (р < 0.001); the spontaneous NBT reduction test showed an increase from 50.7 to 58.6 a.u. (p < 0.01) 30 to 60 min after exposure. The most pronounced change of the microbicidal activity coefficient was observed 2 min after pain modeling, increasing from 1.40 to 1.72 a.u (р < 0.001). By the end of the experiment, the measured parameters approximated their initial values. During the analysis, we accounted for the fact that the neutrophil response to the algogenic stimulus was unfolding in the setting of microbial colonization occurring in newborns.

Autoimmune process markers in insulin-dependent diabetes mellitus

Quantitative and functional parameters of the monocyte and В-cell immunity were assessed in patients with insulin-dependent diabetes over the course of disease. Studies of the monocyte component in subjects predisposed to disease and of the humoral one in those with clinical manifestations are valuable for predicting the autoimmune process. Signs predicting a poor outcome are increase of the count of Fc-positive cells, decrease of NBT reduction of monocytes in subjects without signs of the disease, high levels of immunoglobulin G in manifest disease, and increase of IgM and B-lymphopcyte count and decrease of IgA over the course of the disease.

ASLAN003, a Novel and Potent Dihydroorotate Dehydrogenase (DHODH) Inhibitor, Induces Differentiation of Acute Myeloid Leukemia

Background: Differentiation therapies achieve remarkable success in acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). However, clinical benefits of differentiation therapies are negligible in non-APL AML, which accounts for the majority of AML cases. Dihydroorotate dehydrogenase (DHODH) regulates the fourth step of the de novo pyrimidine synthesis pathway. DHODH is a key therapeutic target for auto-immune diseases and cancer, particularly differentiation of AML. ASLAN003 is a novel, potent small molecule DHODH inhibitor being developed in AML by ASLAN Pharmaceuticals. Methods: We investigated activity of ASLAN003 in AML cell lines and primary bone marrow (BM) cells (NUS Leukemia Tissue Bank) from patients with AML (N = 14) or myelodysplastic syndromes (MDS) (N = 6) and healthy control (N = 1). We performed CTG assay, FACS analysis of cell viability and myeloid markers, wright-giemsa staining, NBT reduction assay, and qRT-PCR analysis of key lineage transcription factors to evaluate the effects of ASLAN003 on cell growth, differentiation, apoptosis, and gene expression changes in vitro. Two AML cell lines and 1 leukemic patient derived xenograft (PDX) line (NUS Leukemia Tissue Bank) were studied in NSG xenograft mice. Mice were administrated with vehicle control or ASLAN003 50 mg/kg by oral gavage once daily. Results: ASLAN003 inhibited leukemic cell growth of THP-1, MOLM-14 and KG-1 with IC50 of 152, 582 and 382 nM, respectively, at 48 h. Treatment of these leukemia cells with ASLAN003 for 96 h consistently resulted in remarkable increase of CD11b (p < 0.001) and displayed morphologic changes of terminal differentiation and positivity for NBT reduction. ASLAN003 was active in differentiation with an EC50 of 28, 85, and 56 nM, in these 3 lines, respectively. ASLAN003 induced approximately 2-fold higher CD11b+ cells than Brequinar (BRQ), another DHODH inhibitor. Addition of uridine rescued differentiation and improved cell viability in ASLAN003 treated-cells, implying on-target specificity of ASLAN003. Mechanistically, ASLAN003 induced differentiation through induction of myeloid lineage transcription factor Runx1, Pu.1, Gif1 and repression of HoxA9, Gata1. The response of primary BM cells to ASLAN003 was classified into 3 categories: sensitive if any of myeloid markers CD11b, CD14, CD13 or CD33 increased ≥ 15%; moderate: ≥ 5%, but < 15%; resistant: < 5%. Among AML samples, we observed 6 (43%) sensitive cases, 6 (43%) moderate cases and 2 (14%) resistant cases. Three (50%) MDS samples displayed sensitive response and 3 cases (50%) showed moderate response. The healthy control sample was resistant to ASLAN003. Importantly, ASLAN003 promoted differentiation and cell death of myeloid cells in one relapsed AML case. Morphologic analysis and NBT assay demonstrated the features of neutrophil differentiation in selected ASLAN003-treated primary AML blasts. For in vivo experiments, significantly prolonged survival was seen in ASLAN003-treated groups when compared to vehicle control group in both MOLM-14 (p = 0.031) and THP-1 (p < 0.001) xenograft models. ASLAN003 substantially reduced disseminated tumors and leukemic infiltration into liver in xenografted mice. The human CD45+ cells were significantly reduced in BM, peripheral blood, spleen and liver, with significantly increased differentiation of AML cells (CD11b and CD14 positive cells) in BM of treated mice in both models (p < 0.01). We also evaluated the therapeutic efficacy of ASLAN003 in one PDX line, AML-14. At the end of experiments (day 77 post treatment), all PDX mice were alive in both control and ASLAN003 group. The leukemic burden was significantly lower in ASLAN003-treated PDXs than in vehicle-treated PDXs (p = 0.04). Overall, these data demonstrate potent in vivo efficacy of ASLAN003 in inducing myeloid differentiation of blast cells and the drug appears highly tolerable even after prolonged administration. Conclusion: ASLAN003 is a novel, highly potent DHODH inhibitor that induces terminal differentiation, inhibits cell growth and promotes cell death of AML blasts, including relapsed AML blasts. ASLAN003 prolongs survival and shows therapeutic effects in mice bearing different AML cell lines and reduces leukemic burden in an AML PDX model. Currently, ASLAN003 efficacy is being evaluated in a Phase IIa clinical trial in patients with AML (NCT03451084; Ting, ASH abstract 2018). Disclosures Seet: ASLAN Pharmaceuticals: Employment, Equity Ownership. Ooi:ASLAN Pharmaceuticals: Employment, Equity Ownership. Lindmark:ASLAN Pharmaceuticals: Employment, Equity Ownership. McHale:ASLAN Pharmaceuticals: Employment, Equity Ownership. Chng:Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Aslan: Research Funding; Merck: Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

Immunomodulatory and antiarthritic activities of Smilax zeylanica

<p><em>Smilax zeylanica </em>Linn has been traditionally used in the treatment of rheumatoid arthritis but, no scientific data has been published supporting the claimed ethanomedical use. This study was designed to investigate the immunomodulatory and antiarthritic activities of <em>Smilax zeylanica</em>.</p><p>Methanolic extract of <em>Smilax zeylanica </em>(MESZ) roots was tested for its immunomodulatory activity by NBT reduction test. Antiarthritic activity of the same was tested by <em>in vitro </em>protein denaturation and <em>in vivo </em>complete Freunds adjuvant (CFA) induced arthritis.<strong> </strong></p><p>MESZ<strong> </strong>showed its significant effect on both cell mediated and humoral immunity to suppress stimulated immune responses in NBT reduction test . It also markedly inhibited protein denaturation in <em>in vitro </em>model. Extract at 200 mg/kg and 400 mg/kg showed statistically significant inhibition ( p&lt;0.05) of the edema formation in CFA model. Histopathological studies of ankle joints also supported this finding.</p>The presence of steroids in the extract might be responsible for the prominent immunomodulatory and antiarthritic activities of the plant. Hence the present study concluded that <em>Smilax zeylanica</em> holds immunomodulatory and antiarthritic activities.

Standardization and regulation of the rate of the superoxide-generating adrenaline autoxidation reaction used for evaluation of pro/antioxidant properties of various materials

The superoxide-generating reaction of adrenaline autoxidation is widely used for determination of the activity of superoxide dismutase and pro/antioxidant properties of various materials. There are two variants of the spectrophotometric registration of the products of this reaction. The first is based on registration of adrenochrome, as adrenaline autooxidation product at 347 nm; the second employs nitro blue tetrazolium (NBT) and registration of diformazan, a product of NBT reduction at 560 nm. In the present work, recommendations for the standardization of the reaction rate in both variants have been proposed. The main approach consists in the use of the pharmaceutical form of 0.1% adrenaline hydrochloride solution. Although each of two adrenaline preparations available in the Russian market has some features in kinetic behavior of its autooxidation; they are applicable in the superoxide generating system based on adrenaline autooxidation. Performing measurements at 560 nm, the reaction rate can be regulated by lowering the concentration of added adrenaline, whereas during spectrophotometric registration at 347 nm, this cannot be done. These features of adrenaline autoxidation may be due to the fact that the intrinsic multistage process of the conversion of adrenaline to adrenochrome, which is recorded at 347 nm, is coupled with the transition of electrons from adrenaline and intermediate products of its oxidation to oxygen, carbon dioxide, and carbonate bicarbonate ions, which is detected in the presence of added NBT.

Reactive Oxygen Intermediate (ROI) in Dog Macrophage Infected with Mycobacterium tuberculosis

experiment used 24 healthy dogs, aged between 1 and 2 years, both male and female which were divided into twodifferent groups consisting of 12 dogs each. The first group was the treatment group, that is they were infected with Mtuberculosis and the second one was the control group. The activity of macrophages ROI secretion were measured at1st, 2nd, 12th, and 24th after infection using nitroblue tetrazolium (NBT) reduction assay. Three cats were used to measure themacrophage activity in each period, using triplicate sample for each cat. The results of the experiment showed thatROI secretion increased in infected group compared with the control group, and this activity reached to the plateaulevel at 2 weeks after infection. Although these enhanced activities were gradually diminished thereafter, higherlevels of these activities were consistently observed until the end of experiment compared with control group. Theresults of the experiment indicated that ROI played an important role to against M.tuberculosis infection in dogs.Keyword: macrophage, ROI, M.tuberculosis, dogs

EKSTRAK ETANOL BIJI JINTAN HITAM (Nigella sativa) MENINGKATKAN AKTIVITAS FAGOSITOSIS MAKROFAG MENCIT SWISS YANG DIINFEKSI Lysteria monocytogenes

Penelitian ini bertujuan mengetahui pengaruh ekstrak etanol biji jintan hitam (EEBJH) terhadap aktivitas fagositosis dan sekresi reactive oxygen intermediates (ROI) makrofag peritoneal mencit Swiss yang diinfeksi Lysteria monocytogenes (L. monoytogenes). Dalam penelitian ini digunakan 72 ekor mencit jantan galur Swiss dengan berat antara 20-30 g. Mencit dibagi ke dalam enam kelompok, masing-masing terdiri atas 12 ekor. Kelompok 1 (kelompok kontrol negatif), diberi akuades secara per oral. Kelompok II (kelompok kontrol positif), hewan uji diberi imboost per oral. Kelompok III, IV, V, dan VI sebagai kelompok perlakuan, masing-masing diberi EEBJH dengan dosis 1, 5, 25, dan 125 mg/kg bobot badan/hari per oral selama 14 hari. Pada hari ke-15, semua mencit diinfeksi L. monocytogenes. Aktivitas fagositosis makrofag peritoneal diamati dengan metode lateks sedangkan aktivitas sekresi ROI diamati dengan metode nitro blue tetrazolium (NBT) reduction assay. Hasil penelitian menunjukkan bahwa pemberian EEBJH meningkatkan aktivitas fagositosis dan sekresi ROI makrofag peritoneal yang diinfeksi L. monocytogenes. Angka kematian hewan uji pada kelompok perlakuan lebih rendah dari kelompok negatif. Aktivitas fagositosis dan sekresi ROI makrofag tertinggi terdapat pada hari ke-14. Berdasarkan hasil penelitian maka dapat disimpulkan bahwa EEBJH memiliki efek meningkatkan aktivitas fagositosis dan sekresi ROI makrofag mencit Swiss yangdiinfeksi L. monocytogenes. Kelompok perlakuan dengan dosis 5 mg/kg bobot badan EEBJH memiliki aktivitas fagositosis dan sekresi ROI tertinggi.

Flavonoid 4′-O-Methylkuwanon E fromMorus albaInduces the Differentiation of THP-1 Human Leukemia Cells

Aims. In this work we studied cytodifferentiation effects of newly characterized prenyl flavonoid 4′-O-methylkuwanon E (4ME) isolated from white mulberry (Morus albaL.).Main Methods. Cell growth and viability were measured by dye exclusion assay; cell cycle and surface antigen CD11b were monitored by flow cytometry. For the cytodifferentiation of cells the NBT reduction assay was employed. Regulatory proteins were assessed by western blotting.Key Findings. 4ME induced dose-dependent growth inhibition of THP-1 cells, which was not accompanied by toxic effect. Inhibition of cells proliferation caused by 4ME was associated with the accumulation in G1 phase and with downregulation of hyperphosphorylated pRb. Treatment with 4ME led to significant induction of NBT-reducing activity of PMA stimulated THP-1 cells and upregulation expression of differentiation-associated surface antigen CD11b. Our results suggest that monocytic differentiation induced by 4ME is connected with up-regulation of p38 kinase activity.Significance. Our study provides the first evidence that 4ME induces the differentiation of THP-1 human monocytic leukemia cells and thus is a potential cytodifferentiating anticancer agent.

Involvement of carbonate/bicarbonate ions in the superoxide-generating reaction of adrenaline autoxidation

An important role of carbonate/bicarbonate ions has been recognized in the superoxide generating reaction of adrenaline autooxidation in an alkaline buffer (a model of quinoid adrenaline oxidation in the body). It is suggested that these ions are directly involved not only in formation of superoxide anion radical (О2 -·) but also other radicals derived from the carbonate/bicarbonate buffer. Using various buffers it was shown that the rate of accumulation of adrenochrome, the end product of adrenaline oxidation, and the rate of О2 -· formation depend on concentration of carbonate/bicarbonate ions in the buffer and that these ions significantly accelerate adrenaline autooxidation thus demonstrating prooxidant properties. The detectable amount of diformazan, the product of nitro blue tetrazolium (NBT) reduction, was significantly higher than the amount of adrenochrome formed; taking into consideration the literature data on О2 -· detection by NBT it is suggested that adrenaline autooxidation is accompanied by one-electron reduction not only of oxygen dissolved in the buffer and responsible for superoxide formation but possible carbon dioxide also dissolved in the buffer as well as carbonate/bicarbonate buffer components leading to formation of corresponding radicals. The plots of the dependence of the inhibition of adrenochrome and diformazan formation on the superoxide dismutase concentration have shown that not only superoxide radicals are formed during adrenaline autooxidation. Since carbonate/bicarbonate ions are known to be universally present in the living nature, their involvement in free radical processes proceeding in the organism is discussed.