Nonspecific Agents Research Articles

Treatment of moderate to severe orbitopathy: Current modalities and perspectives

Graves’ orbitopathy (GO) is the primary cause of exophthalmos in adults. It appears in 30 to 50% of patients with Graves’ disease. About 5% are moderate-to-severe cases that might be see-threatening or lead to long term disabling sequelae. Recommendations have been established in 2016 by the European thyroid association (ETA) and the European group on Grave's orbitopathy (EUGOGO), suggesting a wide use of corticosteroids in moderate to severe forms. However, disappointing results have been reported in 20 to 30% of cases. Improved understanding of pathophysiological mechanisms has allowed the use of non-specific immunomodulatory agents, currently under evaluation, and which place in the therapeutic strategy remains to be determined. Very recently, new promising therapeutic advances have emerged with the identification of new therapeutic targets, such as the TSH receptor and IGF-1 receptor complex.

The Effect of the EGFR - Targeting Compound 3-[(4-Phenylpyrimidin-2-yl) Amino] Benzene-1-Sulfonamide (13f) against Cholangiocarcinoma Cell Lines

Objective:Cholangiocarcinoma (CCA) is a noxious malignancy of epithelium of the bile duct with a low response rate to chemotherapy. The epidermal growth factor receptor (EGFR) signaling pathway is implicated in the development of cancerous cells, especially CCA. In this study, we report detailed biological profiling of 13f identified from our earlier hit expansion studies. The aim of this work was to expand our understanding of 13f via more detailed investigations of its mechanism of action against KKU-100, KKU-452 and KKU-M156 CCA cells, as well as in comparison to the EGFR inhibitor Gefitinib and non-specific chemotherapeutic agents such as Cisplatin. Methods:Inhibiting EGFR-Kinase, cytotoxicity, clonogenic assay, wound healing and apoptosis were performed. Levels of total expression of EGFR and EGFR phosphorylation proteins were detected. Results:13f was confirmed as an inhibitor of EGFR with an IC50 value against the tyrosine kinase of EGFR of 22 nM and IC50 values for 48 h incubation period were 1.3 ± 1.9, 1.5 ± 0.4 and 1.7 ± 1.1 µM of KKU-100, KKU-452 and KKU-M156, respectively through dose- and time-dependent induction of early apoptosis of CCA cells. The compound also suppressed the clonogenic ability of KKU-100 and KKU-M156 cells stronger than Gefitinib, while potently inhibiting EGF-stimulated CCA cell migratory activity in KKU-452 cells. It was observed that under normal conditions EGFR was activated in CCA cells. EGF-stimulated basal expression of EGFR in KKU-452 cells was suppressed following 13f treatment, which was significantly greater than that of the marketed EGFR inhibitor Gefitinib. Conclusion:In summary, our study showed that 13f has potent anti-cancer activities including antiproliferation, clonogenic ability and migration through the modulation of EGFR signaling pathway in CCA for the first time. The compound represents an interesting starting point as a potential chemotherapeutic agent in ongoing efforts to improve response rate in CCA patients.

Molecular Linking of HIPEC (Hyperthermic Intraperitoneal Chemotherapy) and Tregs (Regulatory T- cells) in Advanced Epithelial Ovarian Cancer - A Review

The mainstay in the management of advanced epithelial ovarian cancer is platinum-based chemotherapy and complete cytoreductive surgery. Despite this, about two-thirds of patients have disease recurrence mostly within the peritoneal cavity. HIPEC (Hyperthermic Intraperitoneal Chemotherapy) is a modality that delivers cell-cycle non-specific chemotherapeutic agents along with heat 41°C to 43°C into the peritoneal cavity. HIPEC is done intra-operatively after achieving complete cytoreduction (which means after removal of all the tumor deposits more than 2.5 mm). Ovarian cancer is associated with the frequent finding of tumor-infiltrating lymphocytes in their tissue microenvironment. Especially studies have shown that ovarian cancer evades immune surveillance by higher expression of FOXP3 T cells. HIPEC has been used in the treatment of primary and recurrent tumors. In this review, we discuss how the significance of HIPEC on genetics and immunology of these patients with cancer have provided unique insights into the molecular and cellular basis of Treg cells. Studies of HIPEC and its association with Tregs cells should make it possible to increase the paucity of immuno- therapeutic modalities of most human cancer at an unprecedented level of molecular and cellular precision. The predictive, preventive, and therapeutic implications of these studies of HIPEC in relation to immunity in EOC may extend to patients with other peritoneal carcinomatosis.

Use of direct oral anticoagulants in ICU patients. Part I - Applied pharmacology.

Direct oral anticoagulants (DOACs) have revolutionized the field of anticoagulation in the last decade, mainly due to their fixed dosing, rapid onset of anticoagulant effect, and non-inferiority to vitamin K antagonists for both efficacy and safety. Also, the availability of specific and non-specific reversal agents makes these drugs popular. However, in critically ill patients, significant alterations in drug pharmacokinetics and pharmacodynamics might occur due to various ongoing pathophysiological derangements like decreased gastrointestinal absorption, increased volume of distribution, and impaired renal and/or hepatic function. Insight into these changes is essential regarding the safe and effective use of DOACs in critically ill patients. The current article is anarrative review on the use of DOACs in intensive care unit patients, covering 'Applied pharmacology'; the second article in the series covers 'Clinical evidence'.

1289. Pravibismane is a Potent, Broad Spectrum Anti-Infective Small Molecule that Rapidly Disrupts Bacterial Bioenergetics and Halts Bacterial Growth

BackgroundThe rise in resistance to existing antimicrobials has prompted a need for the development of novel antibiotics. Microbion has identified a novel compound, pravibismane, with potent broad spectrum anti-infective and anti-biofilm activity.MethodsHere we used a variety of assays, including Bacterial Cytological Profiling (BCP), to analyze pravibismane in E.coli to gain insight into its likely mechanism of action (MOA). The BCP profile of pravibismane suggested it rapidly shut down cell growth, potentially by turning off cellular gene or protein expression. This was confirmed using a plasmid based GFP induction assay in E.coli tolC that showed pravibismane strongly reduced expression of GFP. The kinetics, reversibility and MOA of pravibismane was further characterized by using time-lapse microscopy, wash out experiments and measurements of both membrane potential and relative intracellular ATP levels.ResultsWe found that pravibismane acts rapidly (within 30 mins) to completely halt cell growth rather than causing immediate cell lysis such as that observed with non-specific cell damaging agents bleach or detergent. Inhibitor wash out experiments in which cells were exposed to pravibismane for 2 hours, washed to remove the compound, and then observed using time-lapse microscopy revealed that the effect of pravibismane is reversible and that cells recovered 8-12 hrs after removing the compound. Wash out experiments with an E.coli tolC strain carrying a plasmid with an IPTG inducible GFP demonstrated that transcription and translation ultimately resumed in most cells after washout. The bioenergetics of the membrane was measured using DiBAC 4(5), a membrane potential sensitive dye which can enter depolarized cells, which revealed that pravibismane caused depolarization of the membrane within 30 mins of exposure in a concentration dependent manner. Finally, a luciferase assay determined pravibismane reduced ATP levels (resulting in decreased luminescence) within 15 mins of exposure in a concentration dependent manner unlike antibiotic controls that had modest or no effect on luminescence.ConclusionOur results suggest that pravibismane acts rapidly to disrupt cellular bioenergetics, resulting in the immediate cessation of cell growth and protein expression.DisclosuresBrett Baker, M.Sc., D.C., Microbion Corporation (Board Member, Employee)

Use of direct‐acting oral anticoagulants in solid organ transplantation: A systematic review

The use of direct-acting oral anticoagulants (DOACs) has increased secondary to the mounting evidence for comparable efficacy and potentially superior safety to vitamin K antagonists (VKAs) in the general population. However, insufficient data regarding DOAC use in solid organ transplant (SOT) recipients and numerous pharmacokinetic and pharmacodynamic considerations limit their use in this highly selected patient population. A systematic review of recent clinical evidence on the safety and efficacy of DOACs compared to VKAs in SOT recipients was conducted. Additional considerations including transplant-specific strategies for DOAC reversal and common pharmacokinetic/pharmacodynamic concerns were also reviewed. Although current evidence is limited to single-center retrospective analyses, DOACs, especially apixaban, appear to be a safe and effective alternative to VKAs for SOT recipients with stable graft function and without drug-drug interactions. Reliable data on DOAC reversal at the time of transplant surgery are lacking, and clinicians should consider idarucizumab, andexanet alfa, and other non-specific reversal agents on an individual patient basis. There is no evidence supporting deviations from the Food and Drug Administration labeling recommendations for DOAC dosing in the setting of drug-drug interactions, obesity, and renal function, especially in patients on hemodialysis.

Effectiveness of moxibustion therapy in the treatment of urticaria: A protocol for a systematic review and meta-analysis.

Background:Urticaria is a common skin disease in clinic. The main clinical symptoms are sudden attack, various forms, different sizes of wind, and erythema, accompanied by varying degrees of itching. At present, antihistamines, non-specific antiallergic agents, or glucocorticoids are the main treatment, with some side effects and adverse reactions. Moxibustion therapy has shown strong advantages in the treatment of urticaria, and the curative effect is accurate. Therefore, this paper will carry out a systematic evaluation and meta-analysis of the efficacy and safety of moxibustion in the treatment of urticaria.Methods:Eight electronic databases will be searched, including PubMed, Excerpta Medica Database, Web of Science, Cochrane Library, the China National Knowledge Infrastructure (CNKI), Chinese Science and Technology Periodical Database (VIP), Wanfang Database (WF), and Chinese Biomedical Literature Database (CBM). We will search above electronic databases from the beginning to October 2020, without any language restriction, but involving only the human subjects. Clinical efficacy, including total effective rate or cure rate, and recurrence rate will be accepted as the primary outcomes. The itch level, number of clusters, size of clusters, and laboratory test results will be used as secondary outcomes. The Cochrane Handbook of Systematic Review (5.3.0) RCT risk assessment tool will be used to evaluate the risk of bias by 2 independent researchers.Results:After the completion of this study, the results will be reported, so it is not possible to give accurate results at present.Conclusions:The results of this study will provide reliable evidence for the efficacy and safety of moxibustion in the treatment of urticaria.INPLASY Registration number:INPLASY2020100040.

Translesion synthesis of the major nitrogen mustard-induced DNA lesion by human DNA polymerase η.

Nitrogen mustards are among the first modern anticancer chemotherapeutics that are still widely used as non-specific anticancer alkylating agents. While the mechanism of action of mustard drugs involves the generation of DNA interstrand cross-links, the predominant lesions produced by these drugs are nitrogen half-mustard-N7-dG (NHMG) adducts. The bulky major groove lesion NHMG, if left unrepaired, can be bypassed by translesion synthesis (TLS) DNA polymerases. However, studies of the TLS past NHMG have not been reported so far. Here, we present the first synthesis of an oligonucleotide containing a site-specific NHMG. We also report kinetic and structural characterization of human DNA polymerase η (polη) bypassing NHMG. The templating NHMG slows dCTP incorporation ∼130-fold, while it increases the misincorporation frequency ∼10-30-fold, highlighting the promutagenic nature of NHMG. A crystal structure of polη incorporating dCTP opposite NHMG shows a Watson-Crick NHMG:dCTP base pair with a large propeller twist angle. The nitrogen half-mustard moiety fits snugly into an open cleft created by the Arg61-Trp64 loop of polη, suggesting a role of the Arg61-Trp64 loop in accommodating bulky major groove adducts during lesion bypass. Overall, our results presented here to provide first insights into the TLS of the major DNA adduct formed by nitrogen mustard drugs.

Assessment of the hepatic tumor extracellular matrix using elastin-specific molecular magnetic resonance imaging in an experimental rabbit cancer model

To investigate the imaging performance of an elastin-specific molecular magnetic resonance imaging (MRI) probe with respect to the extracellular matrix (ECM) in an experimental hepatic cancer model. Twelve rabbits with hepatic VX2 tumors were examined using 3 T MRI 14, 21, and 28 days after tumor implantation for two subsequent days (gadobutrol, day 1; elastin-specific probe, day 2). The relative enhancement (RE) of segmented tumor regions (central and margin) and the peritumoral matrix was calculated using pre-contrast and delayed-phase T1w sequences. MRI measurements were correlated to histopathology and element-specific and spatially resolved mass spectrometry (MS). Mixed-model analysis was performed to assess the performance of the elastin-specific probe. In comparison to gadobutrol, the elastin probe showed significantly stronger RE, which was pronounced in the tumor margin (day 14–28: P ≤ 0.007). In addition, the elastin probe was superior in discriminating between tumor regions (χ2(4) = 65.87; P < 0.001). MRI-based measurements of the elastin probe significantly correlated with the ex vivo elastinstain (R = .84; P <0 .001) and absolute gadolinium concentrations (ICP-MS: R = .73, P <0 .01). LA-ICP-MS imaging confirmed the colocalization of the elastin-specific probe with elastic fibers. Elastin-specific molecular MRI is superior to non-specific gadolinium-based contrast agents in imaging the ECM of hepatic tumors and the peritumoral tissue.

FEIBA® and Novoseven® Neutralize the Anticoagulant Effects of a Novel Small Molecule FXIa Inhibitor BMS-986177/JNJ-70033093 in Human Plasma and Whole Blood in Vitro

Background: FXIa inhibition is a promising antithrombotic drug target. BMS-986177/JNJ-70033093 (BMS-177/JNJ-3093) is a novel small molecular inhibitor of FXIa currently in Phase II clinical trials with the potential for reduced bleeding risk as compared to the currently approved oral anticoagulantsHowever, reversal of anticoagulation may still be required in patients who have uncontrolled or life-threatening bleeding or need an urgent surgical procedure. Aim: To evaluate the ability of nonspecific reversal agents (NSRAs) FEIBA®, NovoSeven®, Kcentra®, Profilnine®, BeneFix®, Novoeight®, and Cyklokapron® to neutralize the anticoagulation of BMS-177/JNJ-3093 in the activated partial thromboplastin time (aPTT), thromboelastography (TEG) and thrombin generation assay (TGA) in vitro using human plasma or whole blood. Method: aPTT and TEG were performed in human plasma and whole blood, respectively, using standard assay procedures. TGA was performed in human plasma using diluted kaolin aPTT reagent (1:10,000). JNJ-3093 was evaluated at different concentrations (0.3 -10 µM) to cover the anticipated exposures in the Phase II clinical trials. The NSRAs were evaluated at the anticipated concentrations according to the dosing information in their respective labels. Results: BMS-177/JNJ-3093 produced concentration dependent increases in aPTT (up to 4.4x at 10 μM); prolongations of lag time in TEG (2.6X); prolongations of lag time (3X) as well as reductions in peak thrombin generation (70%) in TGA. FEIBA® effectively neutralized the anticoagulant effects of JNJ-3093 in aPTT, TEG and TGA. NovoSeven® neutralized the BMS-177/JNJ-3093-induced prolongations in aPTT, prolongations in lag time in TEG and TGA assays and partially restored the peak thrombin generation in TGA. In contrast, all other NSRAs tested had negligible effects or did not show neutralization of anticoagulation induced by BMS-177/JNJ-3093 in the referenced assays Conclusion: These results demonstrate that FEIBA® and NovoSeven® can effectively neutralize the anticoagulant effects of BMS-177/JNJ-3093 in vitro. A clinical study is required to determine if these agents can reverse the anticoagulant effects of BMS-177/JNJ-3093 in patients. Table Disclosures Bunce: Johnson &amp; Johnson: Current Employment, Current equity holder in publicly-traded company. Huang Devine:Johnson &amp; Johnson: Current Employment, Current equity holder in publicly-traded company. Chintala:Johnson &amp; Johnson: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure: FEIBA: hemophilia A and B patients with inhibitors for: control and prevention of bleeding episodes; use around the time of surgery; routine prophylaxis to prevent or reduce the frequency of bleeding episodes NovoSeven: Treatment of bleeding and prevention of bleeding for surgeries and procedures in adults and children with hemophilia A or B with inhibitors, congenital Factor VII (FVII) deficiency, and Glanzmanns thrombasthenia with a decreased or absent response to platelet transfusions; treatment of bleeding and prevention of bleeding for surgeries and procedures in adults with acquired hemophilia Kcentra: urgent reversal of acquired coagulation factor deficiency induced by vitamin K antagonist therapy in adult patients with need for urgent surgery/invasive procedure or acute major bleeding Profilnine: prevention and control of bleeding in patients with Factor IX deficiency due to hemophilia B BeneFix: control and prevention of bleeding episodes or peri-operative management in adult and pediatric patients with hemophilia B Novoeight: for use in adults and children with hemophilia A for control and prevention of bleeding, perioperative management, and routine prophylaxis to prevent or reduce the frequency of bleeding episodes Cyklokapron: patients with hemophilia for short-term use to reduce or prevent hemorrhage and reduce the need for replacement therapy during and following tooth extraction)

Melatonin attenuates growth factor receptor signaling required for SARS-CoV-2 replication

Melatonin has recently been&#x0D; suggested as a non-specific agent to prevent early (asymptomatic or moderate)&#x0D; infections with SARS-CoV-2 virus, the agent responsible for COVID-19, from&#x0D; developing to severe conditions requiring hospitalization. This recommendation&#x0D; was based on the ability of melatonin to suppress excessive inflammatory&#x0D; reactions and to modulate the immune system by attenuating the innate (blind&#x0D; and potentially harmful) response and potentiating the adaptive (selective and&#x0D; helpful) one. Recent data on the molecular mechanism of COVID-19 infection show&#x0D; that growth factor signaling is required for SARS-CoV-2 replication in the&#x0D; infected cells. When confronted with previously published data on the effects&#x0D; of melatonin on epidermal growth factor signaling, these data strongly suggest&#x0D; that melatonin can also act against the virus itself. Taken together, these&#x0D; data represent an additional argument in favor of using melatonin treatment as&#x0D; both a preventive and curative measure against COVID-19.

Induction of Antigen-Specific Tolerance in T Cell Mediated Diseases.

The development of novel approaches to control unwanted immune responses represents an ambitious goal in the management of a number of clinical conditions, including autoimmunity, autoinflammatory diseases, allergies and replacement therapies, in which the T cell response to self or non-harmful antigens threatens the physiological function of tissues and organs. Current treatments for these conditions rely on the use of non-specific immunosuppressive agents and supportive therapies, which may efficiently dampen inflammation and compensate for organ dysfunction, but they require lifelong treatments not devoid of side effects. These limitations induced researchers to undertake the development of definitive and specific solutions to these disorders: the underlying principle of the novel approaches relies on the idea that empowering the tolerogenic arm of the immune system would restore the immune homeostasis and control the disease. Researchers effort resulted in the development of cell-free strategies, including gene vaccination, protein-based approaches and nanoparticles, and an increasing number of clinical trials tested the ability of adoptive transfer of regulatory cells, including T and myeloid cells. Here we will provide an overview of the most promising approaches currently under development, and we will discuss their potential advantages and limitations. The field is teaching us that the success of these strategies depends primarily on our ability to dampen antigen-specific responses without impairing protective immunity, and to manipulate directly or indirectly the immunomodulatory properties of antigen presenting cells, the ultimate in vivo mediators of tolerance.

Diagnostic and therapeutic evaluation of folate-targeted paclitaxel and vinorelbine encapsulating theranostic liposomes for non-small cell lung cancer

NSCLC is the most common type of lung cancer. However, non-specific contrast agents, radiopharmaceuticals, and treatment methods are insufficient in early diagnosis and eradication of all tumor tissue. Therefore, the formulation of a novel, targeted, specific theranostic agents possess critical importance. In our previous study, paclitaxel and vinorelbine encapsulating, Tc-99m radiolabeled, folate targeted, nanosized liposomes were formulated and found promising due to characterization properties, high cellular uptake, and cytotoxicity. In this study, in vivo therapeutic and diagnostic efficacy of liposomal formulations were tested by biodistribution study, evaluation of tumor growth inhibition, and histopathologic examination after in vitro assays on LLC1 cells. Both actively and passively targeted liposomal formulations exhibited high cellular uptake, and co-drug encapsulating liposomes showed a greater cytotoxicity profiles than free drug combination in LLC1 cells. By the results of biodistribution studies performed in NSCLC tumor-bearing C57BL/6 mice, the uptake of radiolabeled, actively folate targeted, co-drug encapsulating liposomal formulation was found to be higher in tumor tissue when compared to non-actively targeted one. Also, more effective treatment was achieved by using folate-targeted, co-drug encapsulating liposomal formulation when compared to free drugs combination according to changes in tumor size of mice. Furthermore, liposomal formulations showed lower toxicity compared to free drug combinations in the toxicity study considering body weight. Moreover, according to the histopathological study, folate targeted, co-drug encapsulating liposomes not only inhibited the tumor growth effectively but also restricted the lung metastasis entirely.

On-target versus off-target effects of drugs inhibiting the replication of SARS-CoV-2

The current epidemic of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for the development of inhibitors of viral replication. Here, we performed a bioinformatic analysis of published and purported SARS-CoV-2 antivirals including imatinib mesylate that we found to suppress SARS-CoV-2 replication on Vero E6 cells and that, according to the published literature on other coronaviruses is likely to act on-target, as a tyrosine kinase inhibitor. We identified a cluster of SARS-CoV-2 antivirals with characteristics of lysosomotropic agents, meaning that they are lipophilic weak bases capable of penetrating into cells. These agents include cepharentine, chloroquine, chlorpromazine, clemastine, cloperastine, emetine, hydroxychloroquine, haloperidol, ML240, PB28, ponatinib, siramesine, and zotatifin (eFT226) all of which are likely to inhibit SARS-CoV-2 replication by non-specific (off-target) effects, meaning that they probably do not act on their ‘official’ pharmacological targets, but rather interfere with viral replication through non-specific effects on acidophilic organelles including autophagosomes, endosomes, and lysosomes. Imatinib mesylate did not fall into this cluster. In conclusion, we propose a tentative classification of SARS-CoV-2 antivirals into specific (on-target) versus non-specific (off-target) agents based on their physicochemical characteristics.

Reversal Agents for the Direct Factor Xa Inhibitors: Biochemical Mechanisms of Current and Newly Emerging Therapies.

The direct oral anticoagulants targeting coagulation factor Xa or thrombin are widely used as alternatives to vitamin K antagonists in the management of venous thromboembolism and nonvalvular atrial fibrillation. In case of bleeding or emergency surgery, reversal agents are helpful to counteract the anticoagulant therapy and restore hemostasis. While idarucizumab has been established as an antidote for the direct thrombin inhibitor dabigatran, reversal strategies for the direct factor Xa inhibitors have been a focal point in clinical care over the past years. In the absence of specific reversal agents, the off-label use of (activated) prothrombin complex concentrate and recombinant factor VIIa have been suggested as effective treatment options during inhibitor-induced bleeding complications. Meanwhile, several specific reversal agents have been developed. In this review, an overview of the current state of nonspecific and specific reversal agents for the direct factor Xa inhibitors is provided, focusing on the biochemistry and mechanism of action and the preclinical assessment of newly emerging therapies.

DOACs- balance between thrombosis and bleeding - A Review

Bleeding is a common side effect of anticoagulant use. However, the majority of bleeding events are not life-threatening and can be managed conservatively. The first step in managing any significant bleeding event is to temporarily stop using the anticoagulant. The aim of this review was to determine the appropriate management strategy for an acutely bleeding patient on DOACs. Direct oral anticoagulants (DOACs) are now widely used in treatment of venous thromboembolism (VTE) and are recommended first-line over vitamin K antagonists (VKAs) in non-cancer associated VTE. Until recently, supportive measures and infusion of clotting factors were the only available options for reversal of DOACs. Within the last 4 years, approval of specific antidotes has led to hopes for improved outcomes in DOAC-related acute bleeding, however limitations remain including cost, availability and "real-world" data. In severe and life-threatening bleeding events, use of non-specific (e.g. PCC) or specific (e.g. idarucizumab, andexanet alpha) reversal agents are recommended. However, further data is needed to compare outcomes between these two management strategies and identify the cost-effectiveness of these various strategies.

How Do Small Molecule Aggregates Inhibit Enzyme Activity? A Molecular Dynamics Study.

Small molecule compounds which form colloidal aggregates in solution are problematic in early drug discovery; adsorption of the target protein by these aggregates can lead to false positives in inhibition assays. In this work, we probe the molecular basis of this inhibitory mechanism using molecular dynamics simulations. Specifically, we examine in aqueous solution the adsorption of the enzymes β-lactamase and PTP1B onto aggregates of the drug miconazole. In accordance with experiment, molecular dynamics simulations observe formation of miconazole aggregates as well as subsequent association of these aggregates with β-lactamase and PTP1B. When complexed with aggregate, the proteins do not exhibit significant alteration in protein tertiary structure or dynamics on the microsecond time scale of the simulations, but they do indicate persistent occlusion of the protein active site by miconazole molecules. MD simulations further suggest this occlusion can occur via surficial interactions of protein with miconazole but also potentially by envelopment of the protein by miconazole. The heterogeneous polarity of the miconazole aggregate surface seems to underpin its activity as an invasive and nonspecific inhibitory agent. A deeper understanding of these protein/aggregate systems has implications not only for drug design but also for their exploitation as tools in drug delivery and analytical biochemistry.

Management of major bleeding in patients treated with direct oral anticoagulants: from experience to standardized protocols

Evaluation of clinical-laboratory-therapeutic management and related clinical outcomes (thrombotic-hemorrhagic complications) of patients undergoing treatment with direct oral anticoagulants (DOACs) during major bleeding. This is a two-year observational retrospective study. 27 cases of major bleeding in patients undergoing a therapy with DOACs presented to the Emergency Department of Arcispedale Santa Maria Nuova (Reggio Emilia Hospital). 16 cases (59%) underwent reversal of anticoagulation treatment: 19% using specific reversal therapy (idarucizumab) and 81% using non-specific agents [4-factor prothrombin complex concentrate (4F-PCC)]. Routine laboratory data were available for all the cases, but only for some patients it was possible to obtain the plasma dosage of the oral anticoagulant. Laboratory data confirm rapid correction of activated partial thromboplastin time within one hour from the reversal of anticoagulation with idarucizumab. The absence of correlation between standard blood tests and plasma drug dosage in patients treated with factor Xa Inhibitors was confirmed too. The management of major bleeding during treatment with DOACs using reversal therapy (idarucizumab) and non-specific reversal agent (4F-PCC) showed minimal thrombotic (0.3%) and hemorrhagic (0.3%) complications at 90 days; no events occurred after 6 months.

P0517RENAL STEM CELLS (ARPCS) AS A NEPHROPROTECTIVE APPROACH DURING CISPLATIN-INDUCED ACUTE KIDNEY INJURY: A DEFENSE MECHANISM BY EXTRACELLULAR VESICLES CARRYING THE CYP1B1 GENE

Abstract Background and Aims Cisplatin, is a nonspecific cytotoxic agent that primarily interferes with cellular DNA replication and the cell cycle, nevertheless it lacks tumor selectivity and acts also in normal cells. The most serious adverse reaction of cisplatin is Acute Kidney Injury (AKI), limiting its use and efficacy in chemotherapy. Cisplatin nephrotoxicity is observed in more than 30% of older patients, however the mechanism of nephrotoxicity remains unclear and specific preventive measures are not available. Today, there is an urgent need for specific nephroprotective strategies to be used during cisplatin chemotherapy. Recently, we found that tubular stem/progenitor cells (tARPC) are able to protect the tubular epithelial (RPTEC) from cisplatin induced injury, preserving their proliferation and inhibiting apoptosis. The aim of this study was to identify the molecular mechanisms involved in tARPC-mediated resistance to cisplatin. Method Co-cultures of RPTEC cells and tARPCs were exposed to cisplatin (2.5 µM) for 6 h and then kept in culture for 96 h. Gene expression profile was obtained from tARPCs and RPTECs by Agilent SurePrint G3 Human Gene Expression Microarrays. Genespring and R software were used for the analysis. Gene expression data were validated by Real-time PCR. Extracellular vesicles were isolated from cell culture supernatant by miRCURY Exosome Cell/Urine/CSF Kit (Qiagen) and RNA contained in extracellular vesicles was purified, analyzed in quality by Bioanalyzer (RNA nano) and evaluated by qPCR. The BrdU assay and caspase 3 were used to measure proliferation and apoptosis levels. Immunohistochemical expression of activated caspase-3 was used as a marker of apoptosis in RPTECs. Results By a whole-genome gene expression analysis, we found 107 genes specifically modulated by RPTECs in response to cisplatin and, among these, 30 genes induced by ARPCs following the cisplatin damage. In particular, we found a strong upregulation of the CYP1B1 gene (false discovery rate corrected p value &amp;lt;0.05; fold change=4,1). The qPCR confirmed the increase in CYP1B1 levels in the co-cultures with respect to the respective basal conditions (p &amp;lt;0.05). Interestingly, the CYP1B1 mRNA was also enveloped in Extracellular Vesicles released in the cell co-culture media by tARPC and RPTEC after cisplatin exposition. The CYP1B1 gene encodes a member of the cytochrome P450 superfamily of enzymes and the produced enzyme metabolizes procarcinogens, such as polycyclic aromatic hydrocarbons. CYP1B1 has been shown to be active within tumors and is also capable of metabolizing a structurally diverse range of anticancer drugs. It is responsible for the resistance to docetaxel, cisplatin, tamoxifen and nucleoside analogues. CYP1B1 is involved in the detoxification of the body by various exogenous toxic agents, including cisplatin. We found that CYP1B1 gene was expressed at low levels in RPTECs and in cisplatin-damaged RPTECs. Moreover, 96 h days after 2.5 μM exposure to cisplatin, RPTECs reduced the proliferation and underwent in apoptosis, as showed by caspase 3. However, in co-culture with ARPCs, ARPC cellular and extracellular vesicles CYP1B1 gene expression significantly increased, the apoptotic process was stopped and RPTECs increased their proliferation rate. These data support the hypothesis that ARPCs are sensor of cisplatin damaged-RPTEC and confers cisplatin resistance by transferring CYP1B1 gene in extracellular vesicles. Conclusion This is the first evidence of a cisplatin-induced overexpression of CYP1b1 in renal epithelial cells as a defense mechanism against cisplatin toxicity. This is consistent with our previous data showing that renal progenitors are resistant to cisplatin. The findings may have biological and clinical significance in terms of their implications in cellular communications and potential use of CYP1B1 as biomarkers for AKI induced by cisplatin or as protective agent.

Unmet needs in the acute treatment of migraine attacks and the emerging role of calcitonin gene-related peptide receptor antagonists: An integrative review.

Migraine is a prevalent and chronic disease associated with high rates of disability and significant financial and socioeconomic burden. Current acute treatments for migraine attacks include both migraine-specific (e.g., triptans, ergotamines) and nonspecific (e.g., nonsteroidal anti-inflammatory drugs) medications; however, significant unmet treatment needs remain. The authors sought to characterize the nature and drivers of unmet treatment needs in the acute treatment of migraine attacks and describe emerging migraine-specific treatments, that is, calcitonin gene-related peptide (CGRP) receptor antagonists. PubMed searches were conducted using search terms for studies of unmet migraine treatment needs and CGRP receptor antagonists. Additionally, studies presented at recent headache-focused congresses were included. Forty percent of people with migraine report at least 1 unmet treatment need. Many people are unable to use migraine-specific or nonspecific agents because of contraindications, precautions, and tolerability issues. Disease burden (disability, headache severity/frequency) remains high even in those receiving migraine-specific medications. The oral CGRP receptor antagonists, ubrogepant and rimegepant, demonstrated efficacy in reducing migraine pain, migraine-associated symptoms, and disability, with a low adverse event profile, similar to placebo. The availability and use of CGRP receptor antagonists may help reduce the extent of unmet needs in the treatment of migraine attacks, resulting in more patients receiving treatment and better outcomes for people with migraine. Nurse practitioners are well positioned to increase rates of migraine diagnosis/treatment (another key unmet need), using consensus guidelines to guide their approach.