Additive Synergism Research Articles

Synergism of carbonaceous additives in engineering of the supercapacitive performance of α-and λ-phases of manganese oxide

Owing to the presence of valence shells for charge transfer, a high theoretical specific capacitance, and variable redox properties, MnO2 appears as a promising agent in the realm of energy storage. Crystallographic structures of manganese oxide (MnO2), a transition metal oxide, play a crucial role in determining its capacitive behavior by controlling the ion intercalation and double-layer formation. In this work, two different phases of MnO2 were synthesized using the facile chemical reduction method and biological method, say α-MnO2 and λ-MnO2. The phases were incorporated with different carbonaceous additives including GO and CNT while maintaining a constant weight ratio of 8:1:1 between active materials (MnO2), additive, and PVDF binder. Among different composites formed, the best electrode performance is demonstrated by λ-MnO2/CNT/PVDF composite with an excellent specific capacitance of 356 F/g at a scan rate of 1 A/g. Moreover, the best-performing electrodes are investigated with a symmetrical two-electrode system yielding a wide potential window of 1.8V with an outstanding power density of 13.5 KW/Kg at 5 A/g and an energy density of 53.78 Wh/Kg at 1A/g having a specific capacitance of 190 F/g.

Glycyrrhizinic Acid and Phosphatidylcholine Combination as a Preventive Therapy for Experimental Murine Non-Alcoholic Steatohepatitis

Non-alcoholic metabolic-associated steatohepatitis (MASH) is a condition characterized by increasingly high prevalence and incidence, and also represents an important unmet medical need when it comes to effective pharmacotherapy. In this work, we aimed to explore the therapeutic possibilities of the synergistic combined use of glycyrrhizinic acid (GA) and phosphatidylcholine (PC) to prevent experimental MASH. Adult C57Bl/6 mice were used to model dietary/toxic MASH and treated orally by either GA (34.3 mg/kg/d) or a GA + PC combination (34.3 + 158.1 mg/kg/d) for 3 months. Animal locomotion, behaviour, short-term memory, physical performance, neuromuscular joint function, blood biochemistry, and oxidative stress marker levels were evaluated, followed by histological examination of the liver, skeletal muscle and sciatic nerve with tissue ammonia and lipid content determination. Real-time polymerase chain reaction was used to measure the relative expression of several pathogenetic transcript markers. GA and PC showed moderate additive synergism in their anti-inflammatory, antioxidant, hypoammonaemic, hypoglycaemic, and pro-cognitive activities. Differential effects of the agents were seen in regard to anxiety- and depression-like behaviour as well as gene expression. Our results indicate partial pharmacological synergism between GA and PC and validate further research of its potential clinical applications.

Synergistic electrostatic shielding manipulation of Na+ and desolvation effect of Zn2+ enabled by glycerol for long-lifespan and dendrite-free Zn anodes

The development of aqueous Zn-based energy storage devices is severely restricted by uncontrollable Zn dendrite growth, serious parasitic side-reactions, and poor low-temperature performance. In this work, we introduce glycerol (Gl) with an appropriate concentration of 2.1 M (mol L−1) as a synergistic manipulator and desolvater in the 1 M Zn(ClO4)2 and 0.5 M Na2SO4 electrolyte for zinc batteries. Theoretical calculations and experimental analysis confirm that the Gl-Na+ structure adsorbs onto the Zn anode during the plating process, which increases the electrostatic shielding area of negative charge on Zn dendrites and thus inhibit their growth. Meanwhile, the desolvation effect of Zn2+ enabled by Gl promotes the optimization of solvated structure, resulting in uniform deposition of Zn2+. Additionally, Gl suppresses parasitic side-reactions and coagulation of the electrolyte to ensure a homogeneous electrode surface and improve low-temperature performance. In the modified electrolyte, stable Zn plating-stripping has been achieved for 2930 h at 2 mA cm−2 and 1 mA h cm−2, which is about 7.5 times longer than that in the Gl-free electrolyte. Moreover, even at −10 ℃, the hybrid capacitor with activated carbon and V2O5//Zn full battery realizes 95.37% and 62.84% capacity retention after 30,000 and 3000 cycles, respectively. This work presents an effective strategy for the synergism of double additives to promote the stability of the Zn electrode for high-performance aqueous batteries.

Synergism and Antagonism of Anti-Wear Additives as a Method of Confirming the Mechanism of Their Action

Synergism and Antagonism of Anti-Wear Additives as a Method of Confirming the Mechanism of Their Action

Numerical optimization of the BAT-CELL Bio-Ambient-Tests method for engine exhausts toxicity evaluation

The BAT-CELL Bio-Ambient-Tests method is based on the assessment of the influence of the actual toxicity of various types of gas mixtures on living cells, taking into account the additive synergism. Work has been carried out on the application of the BAT-CELL method for testing engine exhaust gases. The application of computational fluid mechanics using Ansys Fluent made it possible to analyse the flow of engine exhaust gases through the aspiration system used, including analysis of shear stress values and their uniformity distribution on the bottom wall of the sampler containing cell culture on the bottom wall of the sampler. The appropriate flow rate of exhaust gases through the aspiration system and the shape of aspiration tubing for the sampler were selected in order to enable uniform contact of gas particles with the cell surface and not to damage them mechanically. The simulation results were verified in real-life tests and confirmed the theoretical assumptions.

Assessing the Single and Combined Toxicity of Chlorantraniliprole with Bacillus thuringiensis against Maize Fall Armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) under Laboratory Conditions

Use of synthetic insecticides for the management of fall armyworm (FAW) Spodoptera frugiperda (J. E. Smith) for a longer period will led to development of insecticide resistance. Identification of an eco-friendly synergistic agent to enhance the toxicity potential and reduced pesticide use as well become mandatory in due process. Hence the present study was formulated to find the single and combined toxicity of chlorantraniliprole and Bacillus thuringiensis (Bt) against the 2nd and 3rd larval instars of S. frugiperda. Single toxicity of chlorantraniliprole against 2nd and 3rd larval instars were 0.87 and 1.52 ppm (LC25); 4.08 and 6.50 ppm (LC50), respectively. With respect to Bt, single toxicity against 2nd and 3rd larval instars were 474.39 and 693.48 ppm (LC25); 1008.62 and 1228.62 ppm (LC50), respectively. Combination effect of chlorantraniliprole with Bt revealed that 2nd instar of FAW showed supplemental synergism at LC50 of chlorantraniliprole + LC25 of Bt. In the case of LC50 of chlorantraniliprole + LC50 of Bt, LC25 of chlorantraniliprole + LC50 of Bt and LC25 of chlorantraniliprole + LC50 of Bt combinations, they showed sub additive synergism. In 3rd instar larvae, the combined toxicity results were similar for all the combinations of chlorantraniliprole + Bt except LC25 of chlorantraniliprole + LC50 of Bt where it showed an antagonistic synergism. Activity of Carboxyl Esterase (CarE), Mixed Function Oxidase (MFO) and Glutathione-S-Transferase (GST) were found to be lesser in chlorantraniliprole LC50 + Bt LC25 combinations than single toxicity treatments. Therefore, combined use of chlorantraniliprole with Bt at LC50 of chlorantraniliprole + LC25 of Bt had supplemental synergism on fall armyworm under laboratory condition.

Synergism interaction between genetic polymorphisms in drug metabolizing enzymes and NSAIDs on upper gastrointestinal haemorrhage: a multicenter case-control study

Background Interindividual genetic variations contribute to differences in patients’ response to drugs as well as to the development of certain disorders. Patients who use non-steroidal anti-inflammatory drugs (NSAIDs) may develop serious gastrointestinal disorders, mainly upper gastrointestinal haemorrhage (UGIH). Studies about the interaction between NSAIDs and genetic variations on the risk of UGIH are scarce. Therefore, we investigated the effect of 16 single nucleotide polymorphisms (SNPs) involved in drug metabolism on the risk of NSAIDs-induced UGIH. Materials and methods We conducted a multicenter case-control study of 326 cases and 748 controls. Participants were sub-grouped into four categories according to NSAID exposure and genetic profile. We estimated odds ratios (ORs) and their 95% confidence intervals (CI) using generalized linear mixed models for dependent binomial variables and then calculated the measures of interaction, synergism index (S), and relative excess risk due to interaction (RERI). We undertook stratified analyses by the type of NSAID (aspirin, non-aspirin). Results We observed an excess risk of UGIH due to an interaction between any NSAID, non-aspirin NSAIDs or aspirin and carrying certain SNPs. The greatest excess risk was observed for carriers of: rs2180314:C>G [any NSAID: S = 3.30 (95%CI: 1.24–8.80), RERI = 4.39 (95%CI: 0.70–8.07); non-aspirin NSAIDs: S = 3.42 (95%CI: 1.12–10.47), RERI = 3.97 (95%CI: 0.44–7.50)], and rs4809957:A>G [any NSAID: S = 2.11 (95%CI: 0.90–4.97), RERI = 3.46 (95%CI: −0.40–7.31)]. Aspirin use by carriers of rs6664:C>T is also associated with increased risk of UGIH [ORaspirin(+),wild-type: 2.22 (95%CI: 0.69–7.17) vs. ORaspirin(+),genetic-variation: 7.72 (95%CI: 2.75–21.68)], yet larger sample size is needed to confirm this observation. Conclusions The joint effect of the SNPs s2180314:C>G and rs4809957:A>G and NSAIDs are more than three times higher than the sum of their individual effects. Personalized prescriptions based on genotyping would permit a better weighing of risks and benefits from NSAID consumption. KEY MESSAGES Multicenter case-control study of the effect of genetic variations involved in drug metabolism on upper gastrointestinal haemorrhage (UGIH) induced by NSAIDs (aspirin and non-aspirin). There is a statistically significant additive synergism interaction between certain genetic polymorphisms and NSAIDs on UGIH: rs2180314:C>G and rs4809957:A>G. The joint effect of each of these single nucleotide polymorphisms and NSAIDs on UGIH is more than three times higher than the sum of their individual effects. Genetic profiling and personalized prescriptions would be useful in managing the risks and benefits associated with NSAIDs.

The Effect of Plant Extracts Kalanchoe daigremontiana and Aloe arborescens on the Metabolism of Human Multiple Myeloma Cells

The effect of plant extracts Kalanchoe daigremontiana, and Aloe arborescens on human multiple myeloma cells' viability was investigated. It was shown that plant extracts of kalanchoe and aloe reduced tumor cells' viability by 13 and 46%, respectively. The combination of plant extracts with doxorubicin showed an additive synergism of action, enhancing the antitumor effect. Treatment of multiple myeloma cells with plant extracts led to a decrease in intracellular reduced glutathione level. The intracellular glutathione level decreased by 25% under the action of kalanchoe extract and by 63% under the action of aloe extract. Extracts from kalanchoe and aloe decreased mitochondrial membrane potential by 19 and 53%, respectively. The results of the study showed that kalanchoe extract increased ATPase activity, but aloe extract did not affect the level of ATPase activity. The results showed that plant extracts of kalanchoe and aloe affect tumor cells' metabolism and contribute to their death. It was concluded that herbal extracts Kalanchoe daigremontiana and Aloe arborescens have antitumor activity, and aloe extract is more effective than kalanchoe.

Abstract B009: The histone deacetylase inhibitor pracinostat and its metabolite SB991 are active as single agents and in combination with 5-azacitidine in acute myeloid leukemia cells

Abstract Background: Pracinostat (SB939) is a class I, II and IV histone deacetylase inhibitor (HDACi), currently in phase 3 in combination with azacitidine for acute myeloid leukemia (NCT03151408). We have reported its strong preclinical activity in lymphomas as a single agent and in combination with several targeted anti-lymphoma agents (AACR 2018, AACR 2019). Here we report the anti-proliferative activities of pracinostat and its primary metabolite SB991 as single agents and in combination with the demethylating agent 5-azacitidine in acute myeloid leukemia (AML) cell lines. Methods. To assess anti-proliferative activity cells were exposed to increasing doses of pracinostat, SB991 (Helsinn) and vorinostat (LC Laboratories) alone or in combination (pracinostat and SB991) with increasing doses of 5-azacitidine (Selleckchem) for 72h, followed by the MTT assay. The Chou-Talalay index was used to determine synergism. Combination indexes (CI) defined the following: additive effect (CI 0.9-1.1), synergism (CI 0.3-0.9) and antagonism (CI > 1.1).For apoptosis and cell cycle analyses cells were treated with 250 nM pracinostat for 72 hours. Results. HL-60 and MV-4-11 cells responded well to pracinostat with IC50 values of 362 nM and 93 nM, respectively. IC50 values obtained for SB991, the main metabolite of pracinostat, were 233 nM and 85 nM for HL60 and MV-4-11, respectively. These IC50 values compared well with those obtained for vorinostat, which equaled 710 nM for HL-60 and 205 nM for MV-4-11. Combination of pracinostat with 5-azacitidine showed synergism in both AML cell lines (CI = 0.51 for HL-60 and CI = 0.69 for MV-4-11). Combination of SB991 with 5-azacitidine achieved synergism in MV-4-11 cells (CI = 0.54) but was not beneficial in HL-60 cells (CI = 1.23). Pracinostat moderately induced apoptosis in HL-60 cells while MV-4-11 cells underwent massive apoptosis. Cell cycle analysis revealed marked G2/M arrest plus moderate accumulation of cells in the sub-G1 phase for HL-60 cells, which was in agreement with the moderate induction of apoptosis measured by Annexin V staining. Also in agreement with the apoptosis analysis, MV-4-11 cells treated with pracinostat all accumulated in the sub-G1 phase. Conclusions. Pracinostat and its main metabolite SB991 exhibited robust anti-proliferative activity as single agents in HL-60 and MV-4-11, comparing favourably with the FDA approved HDACi vorinostat. Pracinostat mediated its anti-proliferative action by inducing apoptosis and G2/M cell cycle arrest. Combination of pracinostat with 5-azacitidine enhanced its activity in AML cells. These results provide pre-clinical rationale for the ongoing phase 3 trials of pracinostat in combination with azacitidine in AML. Citation Format: Afua Adjeiwaa Mensah, Filippo Spriano, Eugenio Gaudio, Annalisa Bonifacio, Emanuela Lovati, Claudio Pietra, Francesco Bertoni. The histone deacetylase inhibitor pracinostat and its metabolite SB991 are active as single agents and in combination with 5-azacitidine in acute myeloid leukemia cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B009. doi:10.1158/1535-7163.TARG-19-B009

EVALUATION OF THE SYSTEMIC AND REGIONAL ANTIBIOTIC THERAPY EFFECTIVENESS AS PART OF COMPLEX THERAPY IN PATIENTS WITH LOCALLY SPREAD BREAST CANCER

In recent years, breast cancer is the most common oncologic pathology and the most common cause of disability among women in developed countries. The aim of the study. To improve direct and long-term results of treatment in patients with locally spread forms of breast cancer (LSBC) by accelerated regression of perifocal inflammatory changes using selective intraarterial application of antibiotics; improving patients’ quality of life. Materials and methods. The main sample consisted of 109 patients.The control group included 65 (61 %) clinical cases of LSBC who were performed series of courses of intravenous systemic polychemotherapy (SPHT) as neoadjuvant therapy accompanied by systemic intravenous antibiotic therapy. The study group consisted of 42 (39 %) patients who were performed selective intraarterial neoadjuvant polychemotherapy course with simultaneous regional use of antibiotic therapy in the intraarterial administration. Results. The regional administration of antibiotics as a part of the complex neoadjuvant therapy, along with the method of selective intraarterial polychemotherapy, has a positive effect on the linear and chronometric regression of perifocal inflammatory changes around the focus of the primary inoperable LSBC, which positively affects the somatic and psychological patient's state and increases the quality of his life. Conclusions. The complex regional impact on the affected organ has a statistically confirmed better effect with bright holistic features, demonstrating the additive synergism of selective techniques.The selective intraarterial antibiotic therapy does not require additional time and material costs while increasing the efficiency of the method. The versatile advanced approach positively affects the somatic and psychological state of the patient.

Studies of the effect of cerebroprotective substances on the course of stress-induced behavioral depression

Introduction. Atrophic disturbances of neurons of the limbic structures of the brain, which lead to insufficient regulation of emotions and mood, cause depression. Substances with cerebroprotective activity have the ability to inhibit further development and even reverse atrophic damage to neurons.
 Materials and methods. Using electrophysiological techniques, the cerebroprotective activity of piracetam, diacamf – (±)-cis-3-(2-benzimidazolyl)-1,2,2-trimethylcyclopentanone-carboxylic acid hydrochloride and the compound R-86, or 3,2’-spiro-pyrrolo-2-oxindole, was investigated in rat hippocampal slices. In behavioral experiments, there was studied the influence of the above substances, which had been administered for 20 days, on the most important manifestations of behavioral depression in rats caused by a five-day swim stress, such as the time of immobilization in the forced swim test and the indicator of preference for consuming sucrose solution. In addition, the influence of piracetam and diacamf was studied on the effects of the classic antidepressant imipramine.
 Results and discussion. It was found that piracetam, diacamf and the compound R-86 in in vitro studies reduced the damage to the pyramidal hippocampal neurons caused by anoxia and aglycemia, the excitotoxic activity of N-methyl-D-aspartate and oxidative stress when hydrogen peroxide was applied to the slices. Cerebroprotective activity of the test substances, when they are systemically administered for 20 days, is linked with their antidepressant-like effect, which was manifested in a decrease in the immobilization time in the swim test and an increase in the sucrose solution consumption indicator. Co-administration of piracetam in rats potentiated antidepressant activity of imipramine, and diacamf showed additive synergism with the antidepressant.
 Conclusion. Substances with cerebroprotective activity in their chronic administration may show an antidepressant-like effect. Those that potentiate the action of classical anidepressants can be used in conjunction with antidepressants during episodes of exacerbation of the disease. Less active cerebroprotective drugs can be recommended during remission for its prolongation.

The effects of small molecule organic additives on the self-assembly and rheology of betaine wormlike micellar fluids

A model zwitterionic surfactant, oleyl amidopropyl betaine (OAPB), that spontaneously forms viscoelastic wormlike micelles in aqueous solution is mixed with a variety of structurally diverse organic additives. By systematically varying the nature of these additives, insight into the effects of their aromaticity and polarity on the bulk assembly and fluid behaviour of these micelles is gained by the complementary use of small-angle neutron scattering and viscosity measurements. Inclusion of non-polar additives causes the wormlike aggregates to transition into microemulsions above a critical additive concentration; the precise partitioning within the micelle is determined using contrast variation. Alternatively, polar additives do not appear to cause evolution from the wormlike structure, but instead influence the fluid rheology, with some serving to significantly increase viscosity above that of the pure surfactant solution. Addition of these molecules is accompanied by an increase in fluid viscosity when the oxygenated group of the additive is resonance stabilised or acidic. This effect is thought to be a result of surfactant–additive synergism, in which charge screening of the surfactant head-groups causes stronger attractions between molecules, increasing the scission energy of the micelles (i.e. reducing their ability to break apart and reform). Further doping of acidic additives past a critical concentration causes phase separation of the wormlike mixtures. According to ultra-small-angle neutron scattering measurements, the incorporation of all additives (polar or non-polar, aromatic or non-aromatic) results in the formation of ‘branched’ wormlike networks. These findings emphasise the significant impact of impurities or additives on the properties of aqueous wormlike micellar systems formed by zwitterionic surfactants, and could also inform selection of solutes for controlling fluid rheology.

Abstract 4400: Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant prostate cancer cells

Abstract Introduction and objectives: It is well known as Warburg effect that anaerobic glycolytic pathway is activated in various type of advanced cancers including prostate cancer (PC). Lactate dehydrogenase-A (LDH-A) controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. Since LDH-A pathways have been implicated in chemoresistance in various cancers, we investigated whether inhibition of LDH-A pathway could mediate the sensitivity to docetaxel (DOC) in human PC cells. Materials and Method: Four PC cell lines (PC3, DU145, LNCaP, LN-CSS ) were used. LN-CSS is one of the LNCaP-derived castration-resistant PC (CRPC) cell lines established in our laboratories. Sodium oxamate (SO) was used as a specific LDH-A inhibitor. The protein expression was detected by western blot analysis using specific antibodies. Cell growth and survival were evaluated by WST-1 assays. Cell cycle progression and apoptotic inducibility were evaluated by flow cytometry using propidium iodide and Annexin V. The cytotoxicity of SO/DOC combination on PC cells was evaluated using the Chou-Talalay combination index (CI) method which offers quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations. Result: Western blot analysis showed that LDH-A protein was highly expressed in LN-CSS cells compared with other PC cell lines including the parental LNCaP. WST-1 assays showed that treatment with SO (50 mM) for 72 hours in PC cells resulted in growth inhibition (PC3; ~30%, DU145; ~55%, LNCaP; ~20%,LN-CSS; ~55% ), while SO has little growth inhibitory effects on normal lymphocytes in the concentrations between 1-100 mM. IC50 to DOC in PC cells were showed to be 4 nM, 1 nM, 1nM, and 4.5 nM in PC3, DU145, LNCaP and LN-CSS, respectively, suggesting that both PC3 and LN-CSS were relatively resistant to DOC compared with DU145 and LNCaP. Synergistic cytotoxicity was observed after the combination therapy with DOC and SO in LN-CSS (CI: 0.5) but not in PC3 (CI: 1.9), DU145 (CI: 2.0), or LNCaP (CI: 6.5). Cell cycle analyses revealed that the combination with DOC and SO for 72 hours resulted in the accumulation of cells in G2-M phase followed by sub-G1 accumulation in LN-CSS cells. Annexin V assays showed that 43% apoptosis was induced by the combination therapy in LN-CSS cells, while only 12% by DOC only in LN-CSS cells. Conclusion: Our results strongly suggest that LDH-A plays an important role in DOC resistance in advanced PC cells and inhibition of LDH-A promotes DOC-sensitivity especially in CRPC cells. Our study may provide valuable information for the future development of targeted therapies in patients with CRPC. Citation Format: Hiroyuki Muramatsu, Makoto Sumitomo, Shingo Morinaga, Hiroshi Saiki, Ikuo Kobayashi, Keishi Kajikawa, Genya Nishikawa, Yoshiharu Kato, Masahito Watanabe, Kent Kanao, Kogenta Nakamura, kazuhiro Yoshikawa. Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4400. doi:10.1158/1538-7445.AM2017-4400

Combination chemoprevention with grape antioxidants

Antioxidant ingredients present in grape have been extensively investigated for their cancer chemopreventive effects. However, much of the work has been done on individual ingredients, especially focusing on resveratrol and quercetin. Phytochemically, whole grape represents a combination of numerous phytonutrients. Limited research has been done on the possible synergistic/additive/antagonistic interactions among the grape constituents. Among these phytochemical constituents of grapes, resveratrol, quercetin, kaempferol, catechin, epicatechin, and anthocyanins (cyanidin and malvidin) constitute more than 70% of the grape polyphenols. Therefore, these have been relatively well studied for their chemopreventive effects against a variety of cancers. While a wealth of information is available individually on cancer chemopreventive/anti-proliferative effects of resveratrol and quercetin, limited information is available regarding the other major constituents of grape. Studies have also suggested that multiple grape antioxidants, when used in combination, alone or with other agents/drugs show synergistic or additive anti-proliferative response. Based on strong rationale emanating from published studies, it seems probable that a combination of multiple grape ingredients alone or together with other agents could impart 'additive synergism' against cancer.

C6-ceramide's effect on KRAS-defined colorectal cancer cells treated with oxaliplatin, 5-fluoruracil with and without cetuximab.

619 Background: Cetuximab (Cet) is beneficial for patients with metastatic KRAS Wild type (WT) colorectal cancer (mCRC) only. C6-Ceramide (C6-Cer) can act synergistically with chemotherapy to induce cancer cell death. The aim of this study was to compare growth inhibition percentage (GIP) of cytostatics 5-fluoruracil (5-FU), oxaliplatin (Ox) and Cet with or without C6-Cer in KRAS WT and KRAS mutant (KRAS Mut) CRC cell lines (SW48 and SW480, respectively). Methods: Both cell lines were incubated with IC50 concentrations of test drugs. Drug concentrations included 0.8µM for 5-FU, 0.04µM for Ox, 25 µg/mL for Cet, and C6-Cer concentrations ranged from 5 to 10 µM. Cell survival was assessed 72h after using 0.4% Trypan Blue. Results: With above mentioned concentrations, C6-Cer’s GIP was 78.3% for SW-480 (vs. 33.33% for SW-48). It was also noted that the addition of C6-Cer to a combination of steady concentrations of Ox, Cet and 5-FU increased GIP with an especially significant effect on SW-480. Addition of 5 and 7.5µM resulted in doubling of GIP (75% and 86.25%, respectively, vs 32.5% of 5-FU + Ox + Cet). The greatest effect was seen when 10µM of C6-Cer was added to the IC50 concentrations of chemotherapeutic agents (using the 25µg/mL concentration of Cet), where GIP increased from 32.5% to 92.5% in SW-480. Same concentration of drugs increased GIP for SW-48 to a similar 93.5%. Conclusions: C6-Cer appears to have direct inhibitory properties, especially on KRAS Mut cells. Additionally when added to Ox, 5-FU and Cet, C6-Cer reversed the apparent insensitivity of KRAS Mut to Cet. Also, the study showed C6-Cer can provide additional synergism to their cytostatic properties in KRAS WT CRC cell lines. The true mechanism of these observations is still undergoing analysis, however the effect of isolated C6-Cer on KRAS Mut raises possibility of a different pathway that could bypass EFGR pathway. We believe the results of this study provide a starting point for clinical studies with C6-Ceramide in patients with relapsing or metastatic KRAS Mut CRC in combination with standard chemotherapy plus molecular target agents hoping they will translate into clinical benefit for this difficult to treat patient population.

Abstract 785: IRAK4 inhibitors display synergistic activity when combined with BTK or PI3K inhibitors in B cell lymphomas

Abstract Signaling mediated by interleukin receptor associated kinase 4 (IRAK4) may play a permissive role in certain B-cell lymphomas. In addition, activating mutations of MYD88, which is upstream of IRAK4, have been identified in various B cell malignancies such as DLBCL and WM. Furthermore, there is recent evidence that IRAK4 mediated signaling may work in conjunction with other pathways such involving Bruton's tyrosine kinase(BTK) and phosphoinositide 3-kinase (PI3K). We have identified novel small molecule inhibitors of IRAK1/4 that may be useful therapeutics for the treatment of B cell lymphomas. Lead compounds LG0224912 and LG0250276 were potent inhibitors of IRAK4 in kinase inhibition assays (IC50 = 0.7 nM and 2.9 nM, respectively). Compounds were found to be selective kinase inhibitors after screening in a panel of 381 kinases. Functional inhibition of the IL-1 pathway was determined in the A549 cell line which expresses high levels of IL-1R. A549 cells were stimulated overnight with interleukin-1 and interleukin-6 (IL-6) concentrations were measured by ELISA in the presence of inhibitors or controls. Both compounds inhibited IL-6 with an IC50<100 nM. The anti-proliferative effects of IRAK4 inhibitors were examined in B cell lymphoma cell lines. We tested cell lines containing activating L265P mutation in MYD88 (OCI-LY3 and MWCL1 cell lines) or wild type MYD88 (OCI-LY19 and U266). In all 4 cell lines, IRAK4 inhibitors demonstrated anti-proliferative activity (IC50s ranged from 300 nM - 10 μM). The compounds had varying degrees of activity, but the activity of each compound was relatively consistent among the 4 cell lines. In contrast to previous reports, the in vitro activities of IRAK4 inhibitors were unaffected by the MYD88 mutation. Additional synergism studies were performed with IRAK4 inhibitors combined with either the BTK inhibitor ibrutinib or the PI3K inhibitors idelalisib and TGR-1202. The anti-proliferative effects of drug combinations were measured and a combination index (CI) was calculated using the Chou-Talalay method. Combination treatment of LG0250276 and ibrutinib resulted in a CI of 0.67 indicating moderate synergism in the OCI-LY19 cell line. Combination treatment of LG0250276 and idelalisib demonstrated even greater synergism with a calculated CI of 0.25 in the OCI-LY19 cell line. Comparable results were observed in combinations of LG0250276 with TGR-1202. Similar results were observed in combination studies with the OCI-LY3 cell line. The results suggest that IRAK4 inhibition may have synergistic effects when combined with BTK or PI3K inhibitors in B cell lymphomas irrespective of the MYD88 mutation status. Citation Format: Eric G. Vajda, Robert Niecestro, Lin Zhi, Keith B. Marschke. IRAK4 inhibitors display synergistic activity when combined with BTK or PI3K inhibitors in B cell lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 785. doi:10.1158/1538-7445.AM2015-785

Simulations suggest pharmacological methods for rescuing long-term potentiation

Congenital cognitive dysfunctions are frequently due to deficits in molecular pathways that underlie the induction or maintenance of synaptic plasticity. For example, Rubinstein–Taybi syndrome (RTS) is due to a mutation in cbp, encoding the histone acetyltransferase CREB-binding protein (CBP). CBP is a transcriptional co-activator for CREB, and induction of CREB-dependent transcription plays a key role in long-term memory (LTM). In animal models of RTS, mutations of cbp impair LTM and late-phase long-term potentiation (LTP). As a step toward exploring plausible intervention strategies to rescue the deficits in LTP, we extended our previous model of LTP induction to describe histone acetylation and simulated LTP impairment due to cbp mutation. Plausible drug effects were simulated by model parameter changes, and many increased LTP. However no parameter variation consistent with a effect of a known drug class fully restored LTP. Thus we examined paired parameter variations consistent with effects of known drugs. A pair that simulated the effects of a phosphodiesterase inhibitor (slowing cAMP degradation) concurrent with a deacetylase inhibitor (prolonging histone acetylation) restored normal LTP. Importantly these paired parameter changes did not alter basal synaptic weight. A pair that simulated the effects of a phosphodiesterase inhibitor and an acetyltransferase activator was similarly effective. For both pairs strong additive synergism was present. The effect of the combination was greater than the summed effect of the separate parameter changes. These results suggest that promoting histone acetylation while simultaneously slowing the degradation of cAMP may constitute a promising strategy for restoring deficits in LTP that may be associated with learning deficits in RTS. More generally these results illustrate how the strategy of combining modeling and empirical studies may provide insights into the design of effective therapies for improving long-term synaptic plasticity and learning associated with cognitive disorders.

Computational Analyses of Synergism in Small Molecular Network Motifs

Cellular functions and responses to stimuli are controlled by complex regulatory networks that comprise a large diversity of molecular components and their interactions. However, achieving an intuitive understanding of the dynamical properties and responses to stimuli of these networks is hampered by their large scale and complexity. To address this issue, analyses of regulatory networks often focus on reduced models that depict distinct, reoccurring connectivity patterns referred to as motifs. Previous modeling studies have begun to characterize the dynamics of small motifs, and to describe ways in which variations in parameters affect their responses to stimuli. The present study investigates how variations in pairs of parameters affect responses in a series of ten common network motifs, identifying concurrent variations that act synergistically (or antagonistically) to alter the responses of the motifs to stimuli. Synergism (or antagonism) was quantified using degrees of nonlinear blending and additive synergism. Simulations identified concurrent variations that maximized synergism, and examined the ways in which it was affected by stimulus protocols and the architecture of a motif. Only a subset of architectures exhibited synergism following paired changes in parameters. The approach was then applied to a model describing interlocked feedback loops governing the synthesis of the CREB1 and CREB2 transcription factors. The effects of motifs on synergism for this biologically realistic model were consistent with those for the abstract models of single motifs. These results have implications for the rational design of combination drug therapies with the potential for synergistic interactions.

Ursolic acid from Agastache mexicana aerial parts produces antinociceptive activity involving TRPV1 receptors, cGMP and a serotonergic synergism

Agastache mexicana is a plant that has long been used in large demands in Mexican folk medicine to treat anxiety, insomnia and pain, among others affections. Chromatographic technique was used to identify ursolic acid (UA), 130.7mg/g and 20.3mg/g, as an antinociceptive active compound identified in ethyl acetate and methanol extracts of A. mexicana aerial parts, respectively. Temporal course curves of the antinociceptive response demonstrated a dose-dependent and significant activity of UA (1 to 100mg/kg, i.p.) with an ED50=2mg/kg in comparison to the efficacy of diclofenac (1 or 30 to 100mg/kg, i.p.), a non-steroidal anti-inflammatory drug, with an ED50=11.56mg/kg. The antinociceptive response consisted in the reduction of abdominal constrictions induced with 1% acetic acid in mice. Similarly, UA at 2mg/kg produced significant antinociception in the intracolonic administration of 0.3% capsaicin (a TRPV1 agonist) in mice. It has been reported the inhibition produced by UA on the calcium‐flux induced by capsaicin on TRPV1 receptor suggesting the antagonistic activity of this receptor. Finally, an ED50=44mg/kg was calculated in the neurogenic and inflammatory nociception induced in the formalin test in rats. The antinociceptive response of UA in the formalin test was not modified in presence of naloxone, flumazenil or l-arginine. Nevertheless, it was reverted in presence of 1-H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, an inhibitor of soluble guanylyl cyclase) and increased in presence of N(G)-l-nitro-arginine methyl ester (l-NAME, inhibitor of nitric oxide synthase), theophylline (inhibitor of phosphodiesterase) and WAY100635 (an antagonist of 5-HT1A receptors). Current results provide evidence that the antinociceptive response of A. mexicana depends in part on the presence of UA. Moreover, this triterpene may exerts its antinociceptive effect mediated by the presence of cGMP and an additive synergism with 5HT1A receptors, but also an antagonistic activity towards TRPV1 receptors may be involved.

The Effects of <i>Ganoderma lucidum</i> on Initial Events Related to the <i>Bacillus Calmette-Guérin</i> Efficacy and Toxicity on High-Risk Uroepithelial Cells: An <i>in Vitro</i> Preliminary Study

A novel prophylactic regimen is demanded for preventing bladder cancer recurrence, because of the high side-effect tolls of conventional adjuvant Bacillus Calmette-Guerin (BCG) immunotherapy, in addition to its only moderate efficacy. In vitro and animal studies have demonstrated the anti-cancer properties of a medicinal mushroom called Ganoderma lucidum (GL). In this study, a pre-malignant human uroepithelial cells (HUC-PC) model was utilized to compare the effectiveness between ethanol extract of GL (GLe) and BCG on interleukin-6 (IL-6) secretion and lactate dehydrogenase (LDH) cytotoxicity. Additionally, parameters relevant to the BCG efficacy and safety, including free soluble fibronectin (FN) and cell-surface glycosaminoglycans (GAGs) levels were tested, following the exposure of GLe to the cells. GLe at 100 μg/ml and BCG at 4.8 × 107 CFU were shown to induce equivalent levels of IL-6, suggesting the potential synergism, while the tested concentrations of GLe were non-cytotoxic. During the initial four hours of GLe exposure, the free FN concentrations in harvested media were significantly reduced that might facilitate the binding of BCG for uroepithelial internalization to enhance BCG efficacy. Furthermore, the cell membrane-bound GAGs levels of HUC-PC cells were significant increased in response to GLe to suggest cellular protection from BCG infection. In summary, current findings suggest the potential additive synergism of GLe with the BCG efficacy, as well as its protective effects, and thus reducing the BCG toxicity.