Multifaceted Mechanism Of Action Research Articles

An on-demand antibacterial hydrogel for precise and rapid elimination of bacterial infection in a murine partial thickness scald burn wound

Burn wounds trigger prolonged inflammation, impair healing, and result in a high mortality rate. The effective management of burn patients requires a targeted approach to regulate the wound microenvironment, maintaining sterility while fostering conditions conducive to healing and functionality. Here, we developed a targeted antibacterial pH/temperature-responsive silver nanoparticle (AgNP) hydrogel triggering the release of silver ions based on changes in the burn wound microenvironment. The delivery system not only exerts a strong antibacterial effect owing to the localization and interfacial interaction of ultrasmall AgNPs against the bacterial surface and deeply embedded cells within the biofilm but also downregulates the bacterial pore-forming genes, thereby overwhelmingly deactivating bacterial responses through a multifaceted mechanism of action. We demonstrate that the application of AgNP hydrogel results in pH-dependent bacterial killing and elimination of over 95 % of pathogens while being non-toxic to mammalian cell viability and migration. Furthermore, the in vivo preclinical burn infection murine model demonstrates the eradication of S. aureus infection leading to a progressive burn repair supported by increased collagen deposition, anti-inflammatory properties, and increased burn angiogenesis. Overall, this strategy provides a targeted approach to addressing drug-resistant chronic burn infections, thereby contributing to improved management of burn injuries.

Evaluating the Efficacy and Safety Profile of Good Morning™ Constipation Drops in a Loperamide-Induced Rat Model

Introduction: Constipation is a prevalent gastrointestinal condition globally, often managed with conventional laxatives that may cause side effects and dependency. Homeopathy, offering a holistic approach through diluted natural substances, aims to stimulate the body's self-healing mechanism. This study evaluates Good Morning™ drops, an innovative homeopathic formulation designed to safely and effectively manage constipation. Using a rat model of Loperamide-induced constipation, we evaluated its safety and efficacy, unveiling promising results.Materials and Methods: A rat model of Loperamide induced constipation was utilized to assess the efficacy and safety of Good Morning™ Constipation Drops. 3 unique formulations were prepared and employed to the animal model in 3 different doses. 72 rats were randomly assigned to 12 groups (3 control groups plus 3 formulations in 3 different doses totalling 9 groups), receiving specific variants or control treatments over the period of 21 days. Parameters including body weight, water intake, stool count, intestinal function, haematology, bleeding and clotting time, blood biochemistry, and organ weight were evaluated. Results and discussion: The results showed that all homeopathic formulations, particularly Variant 2, exhibited significant effects in Loperamide induced constipated rats. Across various parameters, Variant 2 consistently influenced food intake, stool characteristics, and small intestine motility. Safety assessments indicated overall well-being, withVariant 2 demonstrating superior survival outcomes. These findings position Variant 2 as a promising candidate for constipation management, emphasizing its consistent efficacy and safety in the rat model. Conclusion: In conclusion, Good Morning™ Constipation Drops exhibited a promising blend of safety, efficacy, and a multifaceted mechanism of action. The formulation, rich in botanical ingredients, demonstrates high safety with favorable survival rates. Efficacy is evident in improved stool counts, stool weights, Small Intestine Transition percentages, and impacts on lipid profiles and liver enzymes, highlighting its potential as a comprehensive solution for constipation relief.

Role of polyphenols in the management of diabetic complications.

Diabetes Mellitus is an endocrine disorder that will affect, about 693 million adults by 2045 worldwide, (>50% increase from 2017). The conventional treatment of the disease, include the oral hypoglycemic drugs which are given in combination with other drugs and are known to possess various adverse effects like gastrointestinal disturbance, nausea, water retention etc. PURPOSE: Due to the urgent need of combating this disorder without side effects, the alternative and complementary therapies should be explored due to their natural origins and comparable safety. Herbal sources serve as new leads, due to the presence of phytoconstituents with potential therapeutic properties, efficacy and safety. In this review, we tried to summarise the polyphenolic phytoconstituents effective in the treatment of diabetic complications. A systematic literature search was conducted using 4 databases (Google scholar, Pubmed, Scopus, Embase) for the identification of relevant data. Search was performed using various key words such as "diabetes", "polyphenols", "marine sources","anti-diabetic polyphenols". The in vitro studies involving the cell lines used in diabetes and animal models were also considered for inclusion. Additional research papers were identified by reviewing abstracts, scrutinizing reference lists, and reviewing previously published review articles. Polyphenols, a group of phytoconstituents are known worldwide for their tremendous antioxidant potential. So, various research groups have explored their mechanism and therapeutic value in diabetic complications, to improve the insulin sensitivity and glucose metabolism, in controlling the glycemic conditions. Polyphenols exhibit effective therapeutic potential in managing diabetic complications through their multifaceted mechanism of action. They exhibit antioxidative, anti-inflammatory, and anti-glycemic properties, which collectively contribute to their beneficial effects in mitigating diabetic complications. Thus, the inclusion of polyphenols into the diet, may be cosidered as an approach of managing diabetes on long term basis. In this review, we have tried to identify polyphenols effective in diabetes and summarize their mechanism of action along with their potential, for the treatment of diabetic complications.

The potential for development of clinically relevant microbial resistance to rifaximin-α: a narrative review.

Rifaximin-α is a gut-targeted antibiotic indicated for numerous gastrointestinal and liver diseases. Its multifaceted mechanism of action goes beyond direct antimicrobial effects, including alterations in bacterial virulence, cytoprotective effects on host epithelial cells, improvement of impaired intestinal permeability, and reduction of proinflammatory cytokine expression via activation of the pregnane X receptor. Rifaximin-α is virtually non-absorbed, with low systemic drug levels contributing to its excellent safety profile. While there are high concentrations of drug in the colon, low water solubility leads to low colonic drug bioavailability, protecting the gut microbiome. Rifaximin-α appears to be more active in the bile-rich small bowel. Its important biologic effects are largely at sub-inhibitory concentration. Although in vitro testing of clinical isolates from rifaximin recipients has revealed rifaximin-resistant strains in some studies, the risk of emergent rifaximin-α resistance appears to be lower than for many other antibiotics. Rifaximin-α has been used for many years for traveler's diarrhea with no apparent increase in resistance levels in causative pathogens. Further, rifaximin-α retains its efficacy after long-term and recurrent usage in chronic gastrointestinal disorders. There are numerous reasons why the risk of microbial resistance to rifaximin-α may be lower than that for other agents, including low intestinal bioavailability in the aqueous colon, the mechanisms of action of rifaximin-α not requiring inhibitory concentrations of drug, and the low risk of cross transmission of rifaximin-α resistance between bacterial species. Reported emergence of vancomycin-resistant Enterococcus in liver-disease patients maintained on rifaximin needs to be actively studied. Further studies are required to assess the possible correlation between in vitro resistance and rifaximin-α efficacy.

Analysis of the molecular composition of humic substances and their effects on physiological metabolism in maize based on untargeted metabolomics.

Humic substances (HSs), components of plant biostimulants, are known to influence plant physiological processes, nutrient uptake and plant growth, thereby increasing crop yield. However, few studies have focused on the impact of HS on overall plant metabolism, and there is still debate over the connection between HS' structural characteristics and their stimulatory actions. In this study, two different HSs (AHA, Aojia humic acid and SHA, Shandong humic acid) screened in a previous experiment were chosen for foliar spraying, and plant samples were collected on the tenth day after spraying (62 days after germination) to investigate the effects of different HSs on photosynthesis, dry matter accumulation, carbon and nitrogen metabolism and overall metabolism in maize leaf. The results showed different molecular compositions for AHA and SHA and a total of 510 small molecules with significant differences were screened using an ESI-OPLC-MS techno. AHA and SHA exerted different effects on maize growth, with the AHA inducing more effective stimulation than the SHA doing. Untargeted metabolomic analysis revealed that the phospholipid components of maize leaves treated by SHA generally increased significantly than that in the AHA and control treatments. Additionally, both HS-treated maize leaves exhibited different levels of accumulation of trans-zeatin, but SHA treatment significantly decreased the accumulation of zeatin riboside. Compared to CK treatment, AHA treatment resulted in the reorganization of four metabolic pathways: starch and sucrose metabolism, TCA cycle, stilbenes, diarylheptanes, and curcumin biosynthesis, and ABC transport, SHA treatment modified starch and sucrose metabolism and unsaturated fatty acid biosynthesis. These results demonstrate that HSs exert their function through a multifaceted mechanism of action, partially connected to their hormone-like activity but also involving hormoneindependent signaling pathways.

Evaluating the genetic basiss of anti-cancer property of Taxol in Saccharomyces cerevisiae model.

Taxol has been regarded as one of the most successful anti-cancer drugs identified from natural sources to date. Although Taxol is known to sensitize cells by stabilizing microtubules, its ability to cause DNA damage in peripheral blood lymphocytes and to induce oxidative stress and apoptosis indicates that Taxol may have other modes of cytotoxic action. This study focuses on identifying the additional targets of Taxol that may contribute to its multifaceted cell killing property, using Saccharomyces cerevisiae. We show that yeast oxidative stress response mutants (sod1Δ, tsa1Δ and cta1Δ) and DNA damage response mutants (mre11∆, sgs1∆ and sub1∆) are highly sensitive to Taxol. Our results also show that Taxol increases the level of reactive oxygen species (ROS) in yeast oxidative stress response mutant strains. Further, 4',6-Diamidino-2'-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining show that Taxol induces apoptotic features such as nuclear fragmentation and chromatin condensation in DNA repair mutants. On the whole, our results suggest that Taxol's cytotoxic property is attributed to its multifaceted mechanism of action. Yeast S. cerevisiae anti-oxidant and DNA repair gene mutants are sensitive to Taxol compared to wild-type, suggesting yeast model can be used to identify the genetic targets of anti-cancer drugs.

Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

BackgroundPlant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays.ResultsAtr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors.ConclusionsThis study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

Abstract 4544: Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models

Abstract CD37 is a tetraspanin expressed on mature B cells where it orchestrates plasma membrane organization, receptor signaling, cell migration and adhesion. As CD37 is abundantly expressed on many mature B cell-derived malignancies, it represents an attractive target for new antibody therapies. DuoHexaBody®-CD37 is a novel biparatopic bispecific CD37 antibody with an Fc domain engineered to enhance antibody hexamerization upon binding to CD37 on the plasma membrane. DuoHexaBody-CD37 was shown to induce potent complement-dependent cytotoxicity (CDC) of malignant B-cell lines and primary chronic lymphocytic leukemia and non-Hodgkin lymphoma patient samples (Oostindie et al., Blood 2018 132:4170; van der Horst et al., Blood 2018 132:4179). Here, we demonstrate that the DuoHexaBody-CD37 mechanism of action also encompasses FcγR-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Daudi and Raji cells opsonized with DuoHexaBody-CD37 induced FcγRIIa and FcγRIIIa signaling in luciferase reporter assays. In agreement with efficient FcγR engagement, opsonization with DuoHexaBody-CD37 resulted in dose-dependent ADCC and ADCP by human healthy donor peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs), respectively. In a 4-hour Chromium-51 release assay, DuoHexaBody-CD37 induced ADCC in Daudi cells with PMBCs from 11 out of 12 tested donors (average EC50 of 9.87 [±9.98] ng/mL; maximum kill 20.4 [±9.2]%), and in Raji cells with 1 out of 3 donors. Image- and flow cytometry-based assays with MDMs illustrated that in presence of DuoHexaBody-CD37, Daudi cells were efficiently engulfed by effector cells from 3 different donors (average percentage phagocytic macrophages 29.7 ± 7.6%) resulting in target cell depletion (maximum depletion 75.9 [±18.0]%). Potent anti-tumor activity of DuoHexaBody-CD37 in vivo was reported in a screening approach using B-cell lymphoma patient-derived xenograft (PDX) models with single-mouse treatment groups. Two weekly doses of 5 mg/kg DuoHexaBody-CD37 resulted in strong tumor growth inhibition (tumor stasis or tumor regression) in 3/9 models compared to untreated tumors. Follow-up cohort PDX studies with eight mice per group confirmed potent, dose-dependent anti-tumor activity of DuoHexaBody-CD37 at doses as low as 1 mg/kg. In summary, DuoHexaBody-CD37 induces efficient tumor cell kill through CDC, ADCC and ADCP in vitro and shows potent anti-tumor activity in B-cell lymphoma PDX models in vivo. These data further strengthen the rationale for exploring the safety and efficacy of DuoHexaBody-CD37 in B-cell malignancy patients. Citation Format: Laurens P. Kil, Simone C. Oostindie, Kristin Strumane, Hilma J. van der Horst, Berris van Kessel, Marije B. Overdijk, Andreas Lingnau, Marcel Brandhorst, Jeroen van den Brakel, Margaret A. Lindorfer, Ronald P. Taylor, Martine E. Chamuleau, Tuna Mutis, A. Kate Sasser, Janine Schuurman, Paul W. Parren, Frank J. Beurskens, Esther C. Breij. Multifaceted mechanism of action of DuoHexaBody-CD37 involves both complement- and Fc gamma receptor-mediated cytotoxicity in pre-clinical B-cell lymphoma models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4544.

The Use of Harpagophytum Procumbens (Martynia Fragrans) in Rheumatology: a Look through the Prism of Comorbidity (Literature Review)

The objective: is to highlight the scientific achievements of the use of Harpagophytum procumbens in patients with osteoarthritis and other rheumatic diseases, including the aspect of comorbid processes inherent in this pathology.Materials and methods. The authors conducted a research in printed and electronic publications, science research bases, the State Register of Medicinal Products of Ukraine using methods of analysis, comparison and synthesis of information data, as well as the results of their own initial studies of the efficacy of Harpagophytum procumbens in patients with osteoarthritis with phenomena comorbid processes.Results. Materials of scientific researches carried out in different countries of the world testify that the extract of Martynia Fragrans has a multifaceted therapeutic effect of patients with osteoarthritis and age-old comorbid processes due to the properties of this plant like hypotensive, cardiotonic, disaggregant, hypoglycemic, choleretic, which improves the digestive system and kidneys qualities. The indicated properties of drugs with Martynia Fragrans are important in terms of a possible beneficial effect on comorbid diseases of the cardiovascular, digestive, genitourinary systems and diabetes mellitus, which occur in patients with osteoarthritis. A further prospect of clinical research in this direction is presented, which will be in line with the main principles of the «Strategy of the World Health Organization in the field of traditional medicine for 2014–2023».Conclusions. Preparations of Martynia (Harpagophytum procumbens) root have a multifaceted mechanism of action and are effective and safe in the treatment of patients with osteoarthritis, arthritis, mild and moderate pains. Achievements of traditional medicine and the latest studies of the effects of the extracts of Martynia Fragrans on metabolism, other organs and systems provide prospects for their positive impact on comorbid diseases in osteoarthritis and improve the overall results of treatment of such patients, being the prospects for further research.

The Use of Harpagophytum Procumbens (Martynia Fragrans) in Rheumatology: a Look through the Prism of Comorbidity (Literature Review)

The objective: is to highlight the scientific achievements of the use of Harpagophytum procumbens in patients with osteoarthritis and other rheumatic diseases, including the aspect of comorbid processes inherent in this pathology.Materials and methods. The authors conducted a research in printed and electronic publications, science research bases, the State Register of Medicinal Products of Ukraine using methods of analysis, comparison and synthesis of information data, as well as the results of their own initial studies of the efficacy of Harpagophytum procumbens in patients with osteoarthritis with phenomena comorbid processes.Results. Materials of scientific researches carried out in different countries of the world testify that the extract of Martynia Fragrans has a multifaceted therapeutic effect of patients with osteoarthritis and age-old comorbid processes due to the properties of this plant like hypotensive, cardiotonic, disaggregant, hypoglycemic, choleretic, which improves the digestive system and kidneys qualities. The indicated properties of drugs with Martynia Fragrans are important in terms of a possible beneficial effect on comorbid diseases of the cardiovascular, digestive, genitourinary systems and diabetes mellitus, which occur in patients with osteoarthritis. A further prospect of clinical research in this direction is presented, which will be in line with the main principles of the «Strategy of the World Health Organization in the field of traditional medicine for 2014–2023».Conclusions. Preparations of Martynia (Harpagophytum procumbens) root have a multifaceted mechanism of action and are effective and safe in the treatment of patients with osteoarthritis, arthritis, mild and moderate pains. Achievements of traditional medicine and the latest studies of the effects of the extracts of Martynia Fragrans on metabolism, other organs and systems provide prospects for their positive impact on comorbid diseases in osteoarthritis and improve the overall results of treatment of such patients, being the prospects for further research.

Effective killing of bacteria under blue-light irradiation promoted by green synthesized silver nanoparticles loaded on reduced graphene oxide sheets.

Graphene oxide (GO) materials loaded with silver nanoparticles (AgNPs) have drawn considerable attention due to their capacity to efficiently inactivate bacteria though a multifaceted mechanism of action, as well as for presenting a synergetic effect against bacteria when compared to the activity of AgNPs and GO alone. In this investigation, we present an inexpensive and environmentally-friendly method for synthesizing reduced GO sheets coated with silver nanoparticles (AgNPs/r-GO) using a coffee extract solution as a green reducing agent. The physical and chemical properties of the produced materials were extensively characterized by scanning electron microscopy (SEM), field-emission gun transmission electron microscopy (FEG-TEM), ultraviolet and visible absorption (UV-Vis), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), inductively coupled plasma-optical emission spectroscopy (ICP-OES) and ion release determination. The results demonstrated that AgNPs/r-GO composites were successfully produced, revealing the formation of micrometer-sized r-GO sheets decorated by AgNPs of approximately 70nm diameter. Finally, bactericidal and photobactericidal effects of the AgNPs/r-GO composites were tested against Staphylococcus aureus, in which the results showed that the composites presented antimicrobial and photoantimicrobial activities. Moreover, our results demonstrated for the first time, to our knowledge, that an efficient process of bacterial inactivation can be achieved by using AgNPs/r-GO composites under blue light irradiation as a result of three different bacterial killing processes: (i) chemical effect promoted by Ag+ ion release from AgNPs; (ii) photocatalytic activity induced by AgNPs/r-GO composites, enhancing the bacterial photoinactivation due to the excited-Plasmons of the AgNPs when anchored on r-GO; and (iii) photodynamic effect produced by bacterial endogenous photosensitizers under blue-light irradiation. In summary, the present findings demonstrated that AgNPs/r-GO can be obtained by a non-toxic procedure with great potential for biomedical-related applications.

Combined administration of mexidol with known medicines

This review summarizes the available data on the combined administration of mexidol with medicines of different pharmacotherapeutic groups. Mexidol has a multifaceted mechanism of action and exhibits a wide range of pharmacological effects. It enhances therapeutic effects of a variety of drugs in research and clinical settings, boosts the effectiveness of therapy prescribed in accordance with the applicable federal standards and contributes to reducing the severity of complications. Effectiveness data and pathogenetic considerations underpinning combination therapy with mexidol and other drugs suggest that this is a viable approach for treating cerebrovascular and cardiovascular diseases, diseases of the nervous system, open-angle glaucoma and alcohol intoxication as well as a number of other diseases.

Ecteinascidins – new antineoplastic drugs from the depths of the seas

Trabectedin, also known as ecteinascidin 743 (Et-743), is a tetrahydroisoquinoline alkaloid that was originally isolated from the Caribbean sea squirt Ecteinascidia turbinata. Trabectedin is one of the first anticancer drugs of marine origin that has been approved in the European Union. The compound is used to treat patients with advanced soft tissue sarcoma in case of failure of conventional anthracyclines and ifosfamide therapy and in the treatment of recurrent platinum-sensitive ovarian cancer in combination with pegylated liposomal doxorubicin. The mechanism of action of trabectedin is based on interaction with the minor groove of DNA double helix. It triggers a cascade of events that interfere with several transcription factors, DNA binding proteins and DNA repair pathways, resulting in G2/M cell cycle arrest and ultimately apoptosis. Moreover, emerging evidence indicates that Et-743 has dual effects. In addition to induce direct growth inhibition, cell death and differentiation of malignant cells it also affects the tumor microenvironment by reducing the production of key inflammatory mediators. A multifaceted mechanism of action may explain positive results when trabectedin is used in the treatment of cancer that exhibits resistance to conventional chemotherapeutics. The widespread use of ecteinascidins in therapy as well as in clinical trials is, however, limited by the high price of these compounds. Therefore, intensive work is being carried out on the search for synthetic analogues of ecteinascidins with equally high activity against tumor cells.

GM604 regulates developmental neurogenesis pathways and the expression of genes associated with amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) is currently an incurable disease without highly effective pharmacological treatments. The peptide drug GM604 (GM6 or Alirinetide) was developed as a candidate ALS therapy, which has demonstrated safety and good drug-like properties with a favorable pharmacokinetic profile. GM6 is hypothesized to bolster neuron survival through the multi-target regulation of developmental pathways, but mechanisms of action are not fully understood.MethodsThis study used RNA-seq to evaluate transcriptome responses in SH-SY5Y neuroblastoma cells following GM6 treatment (6, 24 and 48 h).ResultsWe identified 2867 protein-coding genes with expression significantly altered by GM6 (FDR < 0.10). Early (6 h) responses included up-regulation of Notch and hedgehog signaling components, with increased expression of developmental genes mediating neurogenesis and axon growth. Prolonged GM6 treatment (24 and 48 h) altered the expression of genes contributing to cell adhesion and the extracellular matrix. GM6 further down-regulated the expression of genes associated with mitochondria, inflammatory responses, mRNA processing and chromatin organization. GM6-increased genes were located near GC-rich motifs interacting with C2H2 zinc finger transcription factors, whereas GM6-decreased genes were located near AT-rich motifs associated with helix-turn-helix homeodomain factors. Such motifs interacted with a diverse network of transcription factors encoded by GM6-regulated genes (STAT3, HOXD11, HES7, GLI1). We identified 77 ALS-associated genes with expression significantly altered by GM6 treatment (FDR < 0.10), which were known to function in neurogenesis, axon guidance and the intrinsic apoptosis pathway.ConclusionsOur findings support the hypothesis that GM6 acts through developmental-stage pathways to influence neuron survival. Gene expression responses were consistent with neurotrophic effects, ECM modulation, and activation of the Notch and hedgehog neurodevelopmental pathways. This multifaceted mechanism of action is unique among existing ALS drug candidates and may be applicable to multiple neurodegenerative diseases.

N-acetylcysteine protects against motor, optomotor and morphological deficits induced by 6-OHDA in zebrafish larvae.

BackgroundParkinson’s disease (PD) is the second most common neurodegenerative disorder. In addition to its highly debilitating motor symptoms, non-motor symptoms may precede their motor counterparts by many years, which may characterize a prodromal phase of PD. A potential pharmacological strategy is to introduce neuroprotective agents at an earlier stage in order to prevent further neuronal death. N-acetylcysteine (NAC) has been used against paracetamol overdose hepatotoxicity by restoring hepatic concentrations of glutathione (GSH), and as a mucolytic in chronic obstructive pulmonary disease by reducing disulfide bonds in mucoproteins. It has been shown to be safe for humans at high doses. More recently, several studies have evidenced that NAC has a multifaceted mechanism of action, presenting indirect antioxidant effect by acting as a GSH precursor, besides its anti-inflammatory and neurotrophic effects. Moreover, NAC modulates glutamate release through activation of the cystine-glutamate antiporter in extra-synaptic astrocytes. Its therapeutic benefits have been demonstrated in clinical trials for several neuropsychiatric conditions but has not been tested in PD models yet.MethodsIn this study, we evaluated the potential of NAC to prevent the damage induced by 6-hydroxydopamine (6-OHDA) on motor, optomotor and morphological parameters in a PD model in larval zebrafish.ResultsNAC was able to prevent the motor deficits (total distance, mean speed, maximum acceleration, absolute turn angle and immobility time), optomotor response impairment and morphological alterations (total length and head length) caused by exposure to 6-OHDA, which reinforce and broaden the relevance of its neuroprotective effects.DiscussionNAC acts in different targets relevant to PD pathophysiology. Further studies and clinical trials are needed to assess this agent as a candidate for prevention and adjunctive treatment of PD.

Abstract A184: Activity of plinabulin in tumor models with Kras mutations

Abstract Kras mutations, particularly at codons 12 and 13, are reported to occur in up to one-third of human cancer cells and are considered to be undruggable targets (Stoize et al, 2015). These mutations are especially prevalent in NSCLC and CRC. Plinabulin is a new chemical entity in Phase 3, with a multi-faceted mechanism of action, including anti-angiogenesis, inhibition of tubulin polymerization and activation of JNK, which is downstream of the Kras pathway. The combination of these vascular mechanisms together with activity downstream from Kras, suggests that plinabulin could express anti-tumor activity in the presence of Kras mutations. The present report summarizes the findings with plinabulin on tumors with Kras mutations (p.12V, p.12D, p.D153V, p.G12A, p.G12C, p.G12D, p.G12V or p.G13D) in vitro and in vivo. In vitro, plinabulin was very potent (IC50 7-33 nM) as a cytotoxic agent against CRC cell lines containing a Kras mutation at p.G13D (LoVo, HCT-15, HCT116) or a BRAF + P53 mutation (HT-29) and against multiple myeloma cell lines (Singh et al, Blood, 2010) with either Nras or p.G12A mutations (IC50&amp;lt;10nM). The active metabolite of irinotecan (SN-38) was less active (2-30 fold) in the CRC cell lines. In vivo, plinabulin was tested as a single agent and in combination with SOC in the CRC Kras mutation murine models LoVo and HCT-15 and the P53/Braf mutant model HT-29, the NSCLC Kras mutation model A549, the multiple myeloma Kras mutation model MM.1S and the breast Kras mutation model MDA-MB-231. As a single agent plinabulin was at most moderately active in these models, with the greatest activity seen in the LoVo, HCT-15, and MDA-MB-231 models (TGI 21-43%); the exception was a marked single agent activity observed in the MM.1S model. However, in all cases plinabulin significantly increased the anti-tumor activity of the standard of care agents (TGI 59-84%, with several CRs and increased tolerability as compared to SOC alone). In summary, plinabulin exhibits a potent anti-tumor activity either in vitro, or in vivo as a single agent or in combination with SOC agents. This activity is observed across several tumor types and mutations, but the spectrum of activity does not extend across all tumor types (e.g. inactive in vivo in Kras mutations for pancreatic cancer or melanoma). These findings are encouraging in terms of clinical studies in Kras mutations in MM, NSCLC, CRC and breast cancers. Citation Format: George Kenneth Lloyd, Lihua Du, Gloria Lee, Jessica Dalsing-Hernandez, Kari Kotlarczyk, Paul Gonzales, Steffan Nawrocki, Jennifer Carew, Lan Huang. Activity of plinabulin in tumor models with Kras mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A184.

New therapeutic option for irritable bowel syndrome: serum-derived bovine immunoglobulin.

Oral prescription medical foods have long been used in hospital settings but are also appropriate therapies for gastrointestinal disorders in outpatient medical practice. Oral serum-derived bovine immunoglobulin/protein isolate (SBI) has been shown in clinical studies to reduce loose stools and improve stool consistency as well as other symptoms (i.e., abdominal pain, bloating, and urgency) in patients with irritable bowel syndrome with diarrhea (IBS-D) and human immunodeficiency virus-associated enteropathy. This case series reports the outcomes of 14 IBS patients who received SBI as an addition to standard of care at an individual physician's clinical practice. The patients: 2 IBS with constipation (IBS-C), 7 IBS-D, 2 mixed diarrhea and constipation IBS (IBS-M) and 3 undefined IBS (IBS-U; also described by some physicians as IBS-Bloating), ranged in age from 22-87 years. SBI (5 g or 10 g daily dose) was added to the patient's current standard care and followed for several weeks to determine if symptoms were improved with the addition of SBI. Overall, 12 of the 14 patients indicated some level of improvement through direct questioning of the patients regarding changes from the prior visit. One IBS-Bloating patient had a resolution of symptoms and two patients (1 IBS-Bloating and 1 IBS-C) discontinued therapy because of insufficient relief. The 12 patients who continued on therapy reported an overall improvement in symptoms with better stool consistency, decreased frequency as well as reductions in abdominal pain, bloating, distention, and incontinence. In most cases, therapeutic effects of SBI were seen within the first four weeks of therapy with continued improvements at subsequent visits. SBI has a multifaceted mechanism of action and may help to manage IBS by providing a distinct protein source required to normalize bowel function, gastrointestinal microbiota, and nutritionally enhance tight junction protein expression between intestinal epithelial cells. SBI as a medical food provides a safe option for patients with IBS-D but may have application in other forms of IBS.

Use of SBI, an Oral Medical Food as an Option for Various Gastrointestinal Disorders

Introduction: Oral medical foods have been used for decades for nutritional support, primarily in institutional settings. It is now possible to utilize prescription medical foods for gastrointestinal (GI) disorders in medical practice. Oral serum-derived bovine immunoglobulin/protein isolate (SBI) has been shown to reduce loose stools, improve stool consistency as well as other symptoms (i.e., abdominal pain, bloating, urgency) in patients with diarrhea predominant irritable bowel syndrome (IBS-D) syndrome and HIV-associated enteropathy. This current assessment followed 18 difficult to treat patients in outpatient medical practice setting using SBI as an addition to standard of care. The population consisted of 18 patients: 14 IBS (2 constipation predominant IBS (IBS-C), 7 IBS-D, 2 Mixed IBS (IBS-M), 3 IBS), 2 Crohn’s disease, 1 J-pouch, and 1 chronic ischemic bowel, and ranged in age from 22-92 years. SBI was added to their current care at a daily dose of either 5 or 10 g and followed for several weeks to determine if treatment helped to address and manage their symptoms. Primary symptoms included abdominal pain, diarrhea, bloating, distention and incontinence. Overall, 16 of the 18 patients showed improved symptoms while 2 patients discontinued therapy. One IBS patient was non-compliant during 8 weeks of therapy and 1 IBS-C patient showed improved bowel movements, but felt the therapy was ineffective and chose to discontinue after 11 weeks of therapy. Of the remaining 16 patients who remained on therapy, they reported an overall improvement in symptoms with improved stool consistency, decreased frequency as well as improvements in abdominal pain, bloating, distention, incontinence, nausea and some reports of improved appetites. Two patients reported a resolution or near resolution of symptoms. In most cases, therapeutic effects of SBI were seen within the first 4 weeks of therapy with continued improvements at subsequent visits. In some cases, the removal of other medications was made given patient response with SBI. GI disorders that result in severe chronic changes in motility can be difficult to treat and many patients fail on conventional drug therapies. The multifaceted mechanism of action and specificity of SBI may help to manage chronic motility problems and other symptoms in these difficult to treat patients. Evidence in animal models has shown that SBI can reduce intra-luminal bacterial products, bind with bacterial lipopolysaccharides, and enhance tight junctions between intestinal epithelial cells. Medical foods are a safe treatment option for patients with IBS and other gastrointestinal disorders. Disclosure - Bryon Petschow, PhD - employee of Entera Health, Inc.

Abstract 2922: Stoichiometric depletion of glutathione by anticancer agent JS-K.

Abstract JS-K is a nitric oxide (NO)-releasing drug of the O2-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models. It is activated for NO release by reaction with glutathione (GSH), a step that consumes an equivalent of GSH, effectively irreversibly. The two equivalents of NO thus released can be oxidized intracellularly, leading to S-nitrosylation and S-glutathionylation of GSH in a cascade of further reactions that consume additional equivalents of GSH and generate GSH disulfide (GSSG). The GSH/GSSG redox couple is the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. Here we show that depletion of GSH on treating U937 leukemia cells with JS-K markedly raises the reduction potential of the cell, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. The data indicate that multiplicative depletion of reduced GSH and deregulation of intracellular redox balance are important steps in the mechanism of JS-K's cytotoxic action. An additional GSH-consuming pathway was found to occur with Double JS-K, an aryl bis(diazeniumdiolate) created with the purpose of doubling the payload of NO. Double JS-K caused pronounced protein glutathionylation, in contrast to its monovalent counterpart. Serving as a bivalent electrophile, Double JS-K irreversibly crosslinked GSH to protein thiols through its aromatic linker. The results provide a previously unrecognized component of the multifaceted mechanism of action of bivalent electrophiles of the arylated diazeniumdiolate class. Citation Format: Ryan J. Holland, Anna E. Maciag, Robert Y.-S. Cheng, Luis G. Rodriguez, Joseph E. Saavedra, Lucy M. Anderson, Larry K. Keefer. Stoichiometric depletion of glutathione by anticancer agent JS-K. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2922. doi:10.1158/1538-7445.AM2013-2922

Abstract 3334: GSTP1-activated nitric oxide-releasing/PARP inhibitor hybrid prodrugs induce cancer cell death through ROS/RNS, DNA damage, ER stress, and apoptosis.

Abstract Poly [ADP-ribose] polymerase (PARP) has been recognized as a potential molecular target in cancer therapy. PARP inhibitors have been evaluated in clinical trials, both in combination with DNA damaging chemotherapy, and also as single agents in patients with BRCA deficiencies. However, there are problems associated with the clinical applicability of PARP inhibitors that include theoretical genotoxicity of these DNA damage repair inhibitors, and increased toxicity in combination with chemotherapy. Therefore targeted delivery of PARP inhibitors selectively to cancer cells would be clinically beneficial. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs developed in our laboratory have proven effective as anticancer agents in a number of in vitro and in vivo models. The lead prodrug JS-K exhibits a multifaceted mechanism of action that includes generation of oxidative/nitrosative stress and DNA damage. We hypothesized that inhibiting PARP may increase effectiveness of JS-K. More importantly, creating a PARP inhibitor prodrug that is activated specifically in the cancer cell could diminish dose-limiting adverse events observed in clinical trials. Using structure-based molecular modeling approaches we have designed and synthesized novel diazeniumdiolate/PARP inhibitor hybrid prodrugs that are activated by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed by cancer cells. This GSTP1-catalyzed activation allows for the selective delivery of nitric oxide and PARP inhibitor simultaneously to the cancer cell. Biological evaluation of the prodrugs in vitro in lung adenocarcinoma and leukemia cells reveals that their mechanism of action involves oxidative/nitrosative stress associated with NO release and depletion of cellular GSH, leading to DNA strand break damage. The compounds induce aryl-mediated crosslinking glutathionylation of cellular thiols that is irreversible, and may lead to observed endoplasmic reticulum (ER) stress. The tumor suppressor p53 pathway is involved in cells bearing wildtype p53. We have also observed the activation of stress kinases p38 and SAPK/JNK. All of the above events led to initiation of apoptosis through both intrinsic and extrinsic pathways. Cancer cells often exhibit specific stress-related phenotypes, caused by insults such as reactive oxygen species, DNA damage, metabolic stress, or ER stress, and agents that further enhance these stresses could be developed as potential therapies. GSTP1-activated diazeniumdiolate/PARP inhibitor hybrid prodrugs selectively induce cancer cell death through ROS/DNA damage and ER stress overload, leading to apoptosis. Citation Format: Anna E. Maciag, Joseph E. Saavedra, Ryan J. Holland, Youseung Kim, Vandana Kumari, Christina E. Luthers, Waheed S. Sehareen, Xinhua Ji, Lucy M. Anderson, Larry K. Keefer. GSTP1-activated nitric oxide-releasing/PARP inhibitor hybrid prodrugs induce cancer cell death through ROS/RNS, DNA damage, ER stress, and apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3334. doi:10.1158/1538-7445.AM2013-3334