Scoring indicators observed daily for 5 days.
\r\n\tThe discovery of Nylon by Wallace Hume Carothers, a Harvard-educated world-renowned organic chemist born in Burlington, IA in 1896, successfully crowned the attempts developed by E.I du Pont de Nemours & Company to investigate the structure of high molecular weight polymers and to synthesize the first synthetic polymeric fibre.
\r\n\tWhen it hit the market, it was in the form of stockings and all the women in the US wanted to get their hands on a pair. Despite the successful launch of Nylon on the synthetic fibre market and the high expectations created by its extraordinary features, the unexpected war events in 1941 diverted the production of the new synthetic fibre almost exclusively on military applications. Parachutes, ropes, bootlaces, fuel tanks, mosquito nets and hammocks absorbed the production of Nylon, which helped to determine the WWII events. When the war ended and production returned to pre-war levels, consumers rushed to the department stores in search of stockings, accessories and high-fashion garments.
\r\n\tEven if the world of high fashion now seems to more appreciate the use of natural fibres, Nylon is one of the most widely used polymers for the production of technical fibres and fabrics, automotive and micromechanical components. The global nylon 6 & 66 market is expected to reach USD 41.13 billion by 2025, by the following growth at 6.1% CAGR owing to the Increasing focus on fuel-efficient and less polluting vehicles.
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\r\n\tThe amazing success story of Nylon still continues. While its wide availability inspired the development of innovative applications, such as the additive manufacturing, on the other hand, proper disposal after use of high amounts of Nylon resin energised the development of efficient recycling methodology, including chemical recycling. Moreover, the production of Nylon precursors from biomass has become desirable due to the depletion of fossil hydrocarbons and to reduce greenhouse gas (GHG) emissions. This unique combination of technical and socio-economic driving forces is one that aims to further promote the development of Nylon as one of the most suitable ""best polymers"" with a low ecological footprint.
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\r\n\tThe aim of this publication is to unveil the relationships between the chemical structure and the outstanding properties of the broad family of polyamides and to describe the most recent use of Nylon in fostering new applications and promoting a culture aware of environmental sustainability.
Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the United States [1]. It is estimated that 22,240 women will be diagnosed with OC, and 14,080 women will die of the disease in 2017 [1]. Ovarian malignancies can be primary (arising from normal structures within the ovary) or secondary (arising from non-ovarian tissue). Approximately 90% of all primary OC are epithelial carcinomas [2]. Epithelial ovarian cancer (EOC) is sensitive to many chemotherapeutic agents, and the current standard treatment consists of cytoreductive surgery followed by chemotherapy with platinum compounds such as cisplatin or carboplatin and a taxane agent such as paclitaxel [3]. A high percentage of patients with advanced EOC however, eventually develop recurrent disease within 3 years and only 10–30% of patients presenting with stage III or IV disease survive 5 years following initial diagnosis [3, 4]. This poor survival rate is mainly due to the development of chemotherapy resistance following several rounds of treatment. In many cases, initial recurrences are platinum-sensitive but the disease eventually becomes platinum-resistant; which is defined as disease progressing within 6 months of platinum-based therapy [4, 5]. Platinum-resistant patients are subsequently limited to non-platinum and non-taxane chemotherapy treatment options such as topotecan, gemcitabine, and pegylated liposomal doxorubicin which have shown moderate therapeutic success [6]. Alternative treatment options for platinum-resistant disease are, therefore, constantly being explored and immunotherapy and targeted agents are increasingly undergoing clinical trials which are showing positive results.
\nThe serendipitous discovery that platinum coordination complexes blocked bacterial replication led to the hypothesis that these complexes could be of great clinical value as anti-tumor agents [7]. Cis-diamminedichloroplatinum II (cisplatin) was the first drug in its class successfully marketed followed by carboplatin and oxaliplatin. All three drugs have similar mechanisms of action. Cisplatin and carboplatin are approved for the treatment of OC; while tumor cell resistance mechanisms to both drugs are similar they differ in their pharmacokinetic and toxicity profiles [8]. Oxaliplatin is highly effective in colorectal cancers because it’s mechanism of action (MOA) is not limited to that of the other platinum compounds [8].
\nCisplatin, the prototype platinum compound, is taken up into cells by passive diffusion or via the active copper transporter 1 (CTR1) [9]. The subsequent activation of cisplatin is mediated by the displacement of chloride atoms by water to form a highly reactive electrophile that targets nucleophilic sites on DNA and DNA-associated proteins. The N-7 guanine base is most susceptible, although the O-6 guanine, N1, N3 adenine, and N3 cytosine are also targeted. Cisplatin DNA interactions result in the formation of both mono- and bifunctional adducts with the latter forming cis-Pt (NH3)2-d(GpG) at twice the rate of cis-Pt (NH3)2-d(ApG). Interstrand crosslinks are not as common. The bulky adducts between DNA and cisplatin can bend the helix and unwind DNA. The critical importance is the recognition of DNA-cisplatin adducts by proteins that either initiate DNA repair by nucleotide excision repair (NER) or inhibit repair through high mobility group (HMG) proteins. Platinum compounds are cell cycle non-specific (CCNS) causing arrest in S/G2 [9].
\nMultiple mechanisms are thought to play a role in tumor cell resistance to cisplatin due to the heterogeneity of the disease. Resistance to cisplatin typically confers resistance to carboplatin, but not to oxaliplatin. Some common mechanisms of tumor cell resistance to cisplatin in OC includes increased repair to damaged DNA [10], drug efflux by copper efflux transporters ATP7A [11] and ATP7B [12], reduced uptake by CTR1 [13], and increased expression of glutathione and GSH-S-transferase, which are electron donors forming conjugates with cisplatin and rendering it inactive [10]. Both increased efflux and reduced uptake result in reduced drug accumulation. Overexpression of epidermal growth factor (EGF) and its receptor (EGFR) in cancer cells are critical for growth and survival and EGFR overactivity using autocrine and/or paracrine signals is associated with platinum resistance [14]. The overexpression of the tyrosine kinase; focal adhesion kinase, has also been linked to platinum resistance in OC through several mechanisms including increased expression of the transcription factor OCT4 and the cell surface protein N-cadherin, as well as increased aldehyde dehydrogenase (ALDH) activity [15, 16].
\nAlthough there are many chemotherapeutic agents available, the level of response of platinum-resistant ovarian cancer (OC-Pt) to these drugs is increasingly diminished as the disease progresses [17]. In the past decade, this has fueled a consistent increase in the development of targeted therapies aimed at either supplementing chemotherapeutic regimens or providing novel monotherapy in OC-Pt [17]. Categories of targeted drugs that are undergoing clinical trials or have received FDA approval for OC-Pt include focal adhesion kinase (FAK) inhibitors, poly(adenosine diphosphate [ADP] ribose) polymerase (PARP) inhibitors, anti-angiogenic agents, epidermal growth factor receptor targeting agents, folate receptor antagonists, and insulin growth factor receptor inhibitors.
\nPARP inhibitors are a group of targeted drugs that have been at the forefront of emerging OC-Pt therapeutics over the past decade [18, 19]. Human PARPs comprise a total of 17 enzymes [20]. The PARP-1 isoform was the first member of the family to be described and it is the major active PARP enzyme in human cells with the remainder of activity mainly attributed to the PARP-2 isoform [21]. Both PARP-1 and PARP-2 are DNA damage repair enzymes [21]. Human PARP-1 (113 kDa) is a nuclear protein/enzyme which binds with DNA and promotes DNA repair by releasing PARP-1 from DNA and allows recruitment of proteins involved in both base excisional repair (BER) and homologous recombination [22]. Human PARP-2 (62 kDa) is a nuclear protein that binds less efficiently to DNA single-strand breaks but instead recognizes gaps and flap structures [23]. These DNA repair properties of PARPs have made them important anticancer targets in a variety of cancers including OC.
\nThe inhibition of PARP enzymes, especially PARP-1, results in an excess of single-strand breaks, which subsequently causes double-strand breaks to occur as DNA replicates [24]. Under normal circumstances, defects such as double-strand breaks are usually repaired by the homologous recombination process that involves breast cancer type susceptibility (BRCA) proteins. Tumors with defective homologous recombination, including BRCA1/2-mutated OCs, are therefore very sensitive to PARP inhibition [25].
\nPARP inhibitor drugs are able to cause cancer cell death by inhibiting repair of single-strand breaks and subsequently trapping PARP on DNA, forming cytotoxic PARP-DNA complexes [25]. Several small molecular PARP inhibitor drugs are now undergoing clinical trials and two of them (olaparib and rucaparib) have already been approved by the FDA for use in OC-Pt.
\nOlaparib (Lynparza), a product of AstraZeneca, received approval from the U.S. Food and Drug Administration (FDA) in December 2014. Olaparib is an inhibitor of several PARP enzymes, including PARP1, PARP2, and PARP3 [26]. The orally administered drug is used for monotherapy in patients with germline BRCA-mutated advanced recurrent OC-Pt [26]. Phase II clinical trials have shown that olaparib significantly improves progression-free survival (PFS) in OC-Pt with similar rates of response reported in patients with BRCA1- and BRCA2-mutated disease [26]. The most common side effects observed with olaparib were mild gastrointestinal irritation, anemia, and severe fatigue.
\nRucaparib (Rubraca), a product of Clovis Oncology, was granted accelerated approval from the FDA on December 19, 2016 for the treatment of patients with deleterious BRCA mutation (germline and/or somatic) associated with advanced OC, which had been treated with two or more chemotherapies that included those with OC-Pt. Rucaparib is also a non-specific inhibitor of several PARP enzymes, including PARP1, PARP2, and PARP3 [27]. The ARIEL2 and Study 10 clinical trials produced critical integrated efficacy and safety data in OC-Pt patients which showed that the average response rate was approximately 25% with minimal differences between patients who harbored a BRCA1 mutation, and those who harbored a BRCA2 mutation [27]. Adverse reactions to the drug included fatigue, anemia, dysgeusia, and decreased appetite [27].
\nA third PARP inhibitor niraparib (Zejula), a product of Tesaro, was approved on March 27, 2017 to maintain treatment of adult patients with recurrent epithelial ovarian and fallopian tube cancer that is completely or partially responsive to platinum-based chemotherapy. Niraparib inhibits both PARP1 and PARP2 and currently has no specific indications in OC-Pt [28].
\nIt is generally accepted that the major categories of cancers that are sensitive to PARP inhibitors are BRCA-mutated cancers. Interestingly, drug resistance to PARP inhibitors have been linked to the development of secondary mutations in the BRCA gene themselves [29]. These secondary mutations can restore functional BRCA1 or BRCA2 genes leading to deleterious consequences in patients with cancer [29]. Other mechanisms of resistance to PARP inhibitors include increased multi drug resistance protein-1 (MDR-1) activity, which leads to increased drug efflux from cancer cells as well as reduced expression of tumor suppressor p53-binding protein 1 (TP53BP1), which is required for non-homologous end-joining DNA repair [30]. Many of these resistance mechanisms are active in OC-Pt [10, 11, 12, 13, 14, 15, 16] and therefore can potentially circumvent the therapeutic effects of PARP inhibitors. Nonetheless, PARP inhibitors show much promise in OC-Pt therapeutics.
\nSolid tumors rely on neovascularization for growth and survival in hypoxic environments. The process of angiogenesis is critical for normal ovarian function and for growth, development, and metastasis of OC cells [31]. The hypoxic environment drives angiogenesis in solid tumors which requires continual and persistent growth of new blood vessels [32]. Data strongly suggest a close correlation between increased levels of hypoxia-inducible factor 1-α (HIF 1-α); a transcription factor stabilized during hypoxia and vascular endothelial growth factor (VEGF) in EOC [33]. VEGF is a potent pro-angiogenic growth factor that is upregulated during hypoxia and is elevated in epithelial ovarian neoplasms [33]. VEGF-A is a major pro-angiogenic growth factor that binds to VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2), although VEGFR2 is considered the major target. The VEGF-A/VEGFR-2 interaction activates the RAF/MAPK and PI3K/AKT signaling pathways favoring both proliferation and survival of endothelial cells. Intratumoral protein levels of VEGFR-2 were found to be significantly higher in platinum-resistant OC compared to platinum-sensitive OC patient tumors [34]. Many agents targeting angiogenesis have been developed and several have shown some degree of clinical efficacy in OC-Pt. The anti-angiogenic group of drugs include bevacizumab, aflibercept, nintedanib, trebananib, pazopanib, sunitinib, sorafenib, and cediranib.
\nBevacizumab (Avastin), a monoclonal antibody that binds to the vascular endothelial growth factor (VEGF)-receptor ligand VEGF-A, is the most extensively investigated anti-angiogenic agent in clinical OC research. Currently, it is the only anti-angiogenic drug that is FDA approved for the treatment of OC as monotherapy or in combination regimens with paclitaxel, topotecan, doxorubicin (pegylated), carboplatin, or gemcitabine for recurrent OC-Pt [35]. Bevacizumab potentiates the cytotoxic effect of chemotherapeutic agents by reducing interstitial fluid pressure and vascular permeability to increase delivery of cytotoxic drugs to cancer cells [35].
\nA phase II trial of bevacizumab as a single agent in OC-Pt reported that 40.3% of these patients survived progression free for at least 6 months while median PFS and overall survival were 4.7 and 17 months, respectively [36]. Common adverse effects related to bevacizumab were hematologic and gastrointestinal [36].
\nSubsequent randomized phase III clinical trials focused on the use of bevacizumab with standard chemotherapeutic regimens as first-line treatment in both platinum-sensitive and platinum-resistant OC. AURELIA was the first randomized phase III trial (Study ID#: NCT00976911) to evaluate combined bevacizumab with chemotherapy in OC-Pt [37]. All patients received standard chemotherapy with either paclitaxel or topotecan or liposomal doxorubicin. Patients randomized to arm 2 of the study received bevacizumab (10 mg/kgIV every 2 weeks or 15 mg/kg IV every 3 weeks) concomitantly. The study showed improved PFS and overall response rate with no new safety concerns. The percentage of adverse events associated with chemotherapy + bevacizumab was 57.0% versus 40.3% (chemotherapy alone). Proteinuria and hypertension had the highest incidence rate, whereas gastrointestinal perforations were comparable 2% (bevacizumab) versus 0% (bevacizumab + chemotherapy). Treatment arms that consisted of a higher exposure to chemotherapy in the bevacizumab + chemotherapy combined study group, had a higher incidence rate of hand-foot syndrome and peripheral sensory neuropathy.
\nThe topoisomerase I inhibitor Irinotecan (Camptosar), in combination with bevacizumab was evaluated in recurrent OC in an open-label randomized phase III trial (Study ID#: NCT01091259) [38]. This cohort included 19 patients with OC-Pt. The objective response rate for all patients entered was 27.6% and the clinical benefit rate was 72.4%. Adverse events with the addition of bevacizumab relative to GI toxicity was limited to <3% and considered acceptable [38]. These studies show that it is clinically proven that bevacizumab + chemotherapy demonstrate efficacy in OC-Pt and that safety can be achieved with the right dose and combination of drugs.
\nPazopanib (Votrient) is an oral anti-angiogenic multi-targeted tyrosine kinase inhibitor with activity against VEGFR-1, 2, and 3. Pazopanib is currently FDA approved for advanced renal cell carcinoma and soft tissue carcinoma. The PACOVAR study (Study ID#: NCT01238770) evaluated pazopanib in combination with metronomic cyclophosphamide in 16 patients with platinum-resistant EOC [39]. Metronomic chemotherapy is the close, regular administration of chemotherapy drugs at low, minimally toxic doses, with no prolonged break periods. In the PACOVAR study, median PFS and overall survival were 8.35 and 24.95 months, respectively. The most common adverse events were elevation of liver enzymes, leukopenia, diarrhea, and fatigue. Altogether, five serious adverse events developed in four patients. The study concluded that pazopanib + metronomic cyclophosphamide was a feasible regimen for patients with recurrent OC-Pt.
\nPazopanib has also shown promising results in mice injected with a highly aggressive cisplatin-resistant SKOV-3 clone of OC cells in combination with metronomic oral topotecan (toperisomerase I inhibitor) [40].
\nAflibercept (Ziv-aflibercept/VEGF-trap) mimics the VEGF receptor and has similar ligand binding components to VEGFR-1 and VEGFR-2 [41]. Aflibercept binds to circulating VEGFs and acts like a “VEGF trap” [42]. This primarily results in suppression of VEGF-A and VEGF-B activity and subsequently inhibits the growth of new blood vessels in tumors [42]. Aflibercept was administered at two doses in a randomized, double-blind, phase II trial that assessed response evaluation criteria in solid tumor response rates, as a single agent treatment in recurrent OC-Pt (Study ID#: NCT00327171). The study concluded that the treatment was well tolerated by the patients but the required objective response rate endpoint was not achieved [43]. The participants in this study had received 3–4 prior chemotherapy lines and were resistant to liposomal doxorubicin or topotecan. Hypertension was the most common toxicity observed.
\nFocal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that is encoded by the protein tyrosine kinase 2 (PTK2) gene, and is found in most tissues in the human body [44]. PTK2 gene amplification with subsequent increased activation through phosphorylation occurs in many OCs, where it is involved in promoting cancer cell migration, invasion, adhesion, proliferation, and survival [45, 46, 47]. High FAK activity is generally associated with worse overall cancer patient survival [48, 49]. Several studies have shown that FAK expression is significantly increased in OC-Pt, and that this platinum resistance is associated with increased tumor-associated aldehyde dehydrogenase (ALDH) activity, as well as overexpression of X-linked inhibitor of apoptosis (XIAP) [16, 50]. We have also demonstrated in our studies that platinum-resistant OC cells are resensitized to cisplatin when co-treated with a FAK inhibitor [15].
\nSeveral FAK inhibitors have been developed to prevent FAK activation by blocking its phosphorylation sites; which halts its downstream signaling pathways with subsequent reduction in ovarian tumorigenesis and cancer progression. A few of these drugs are now in clinical trials. The FAK inhibitor defactinib from Verastem was evaluated in a phase I study (Study ID#: NCT00787033) which found that OC-Pt patients achieved a prolonged PFS [51]. Defactinib produced grade 1–2 adverse events that were easily managed and reversible, even with continued dosing [51]. A phase I/Ib, open-label (Study ID#: NCT01778803) multi-center, dose-escalation trial of paclitaxel in combination with defactinib was subsequently initiated in OC-Pt patients with advanced cancers [52]. The combination was found to be efficacious with no apparent increase in the severity and incidence of paclitaxel-related toxicities.
\nA phase I/Ib, open-label, multi-center, dose-escalation, and dose expansion trial (Study ID#: NCT02943317) to evaluate the safety, efficacy, pharmacokinetics, and pharmacodynamics of defactinib in combination with the human monoclonal PD-L1 antibody avelumab in recurrent or refractory stage III–IV OC is currently ongoing, and is expected to enroll approximately 100 patients at up to 15 sites across the United States. The FAK inhibitor GSK2256098 was also evaluated in a phase I clinical trial (Study ID#: NCT01138033) in patients with advanced solid tumors including OC-Pt [53]. GSK2256098 significantly reduced FAK activity in tumors of patients that received the drug at a dose of 750 mg twice daily.
\nFAK inhibition is still an emerging area in OC-Pt therapeutics and many clinical trials are underway that will provide more insight into their efficacy in different histological types of OC.
\nFolate receptors (FRs) are proteins that bind folate with high affinity. The FR-α and FR-β isoforms are well characterized as membrane-bound receptors that facilitate the binding and subsequent internalization of folate compounds and their chemical derivatives [54]. The FR-α receptor is significantly overexpressed in EOC where it promotes tumor growth by either an aberrant folate uptake mechanism or dysregulated signaling pathways [55]. The FR-α receptor can also induce platinum resistance by regulating the expression of apoptosis-related molecules; Bcl-2 and Bax and a higher expression of FR-α level has been linked to poor prognosis in OC patients [56]. These properties of the FR-α receptor makes it a prime therapeutic target for OC. In recent years, two drugs (vintafolide and farletuzumab) have gained relevance as FR-α receptor antagonist applicable in OC-Pt. Farletuzumab (MORAb-003), a monoclonal antibody to FR-α was evaluated in a phase III trial (Study ID#: NCT00738699) in combination with paclitaxel for advanced OC-Pt patients [57]. The drug was developed by Morphotek and the study was unfortunately discontinued because of minimal changes in PFS and the occurrence of serious adverse events including neutropenia and atrial fibrillation [57].
\nVintafolide (originally known as EC145), is a water-soluble derivative of folic acid that is conjugated to the vinca alkaloid ‘desacetylvinblastine hydrazide’ [58]. The combination of vintafolide with pegylated liposomal doxorubicin (PLD) produced a statistically significant increase in PFS for OC-Pt patients [59]. This result was the outcome of the PRECEDENT trial; a randomized phase II study, that compared the combination of vintafolide + PLD with PLD alone [59]. Patients with FR positive cancer showed improved PFS compared to no PFS benefits in FR negative patients. After this successful phase II trial, a phase III trial called the PROCEED study was initiated (Study ID#: NCT01170650) to further evaluate the efficacy and safety of the vintafolide + PLD (Doxil) combination in OC-Pt patients. The main goal of the study is to determine PFS using version 1.1 of the response evaluation criteria in solid tumor (RECIST), and etarfolatide imaging to determine patients FR status [55]. Etarfolatide is a non-invasive, folate receptor-targeting companion imaging agent, which consists of a small molecule targeting the folate receptor and an imaging agent, which is based on technetium-99 m [55].
\nThe targeting of the FR receptor appears to be promising strategy for OC-Pt cancer subsets that significantly overexpress these receptors. New folate conjugates are in development and this area of therapeutics is expected to consistently improve.
\nThe insulin-like growth factor (IGF) system consists of IGF-I, IGF-II, their target receptors (IGF-IR, IGF-IIR, insulin receptor (IR), and the insulin-related receptor (IRR)) as well as a family of six different IGF-binding proteins (IGFBPs) [60]. Upon binding of IGFs to IGF-1R and IR (but not IRR and IGF-2R), many signaling pathways can be activated. These downstream signaling mechanisms include the Ras-Raf-MAPK and PI3K-Akt transduction pathways. These transduction mechanisms result in stimulation of cell proliferation, motility, and inhibition of apoptosis [60]. All IGF-signaling system components are expressed in OC and likewise stimulate cell proliferation, invasive, and angiogenic activity of OC cells [61]. More importantly, IGF-1R/IR inhibition in platinum-resistant ovarian cancer cells resensitizes them to the cytotoxic effects of cisplatin; indicating a role of the IGF system in OC-Pt [62]. This highlights a therapeutic opportunity for insulin and insulin-like growth factor receptor inhibition.
\nIn the past few years, a number of inhibitors targeting the IGFR/IR have been developed, including antibodies against the receptors and small molecule receptor kinase inhibitors [63]. A trial (Study ID#: NCT01708161) with ganitumab (developed by Amgen), a human monoclonal antibody against IGF-IR, has been completed in patients with solid tumors including OC-Pts. This was a multi-center, open-label, phase Ib/II study. The aim of the phase Ib arm, was to estimate the median toxic doses and/or identify the recommended phase II dose(s) for the combination of BYL719 (a PI3K inhibitor) and ganitumab [64]. The phase II arm assessed the clinical efficacy and safety of the combination in OC patient populations including PIK3CA-mutated or -amplified OCs [64]. Data from this study are yet to be released, but will provide insight on the effect of ganitumab in OC-Pt.
\nA phase I/II trial (Study ID#: NCT00889382) with the small molecule, dual IGF-1R/IR tyrosine kinase inhibitor linsitinib (OSI-906) has also been completed [65]. The study evaluated intermittent and continuous linsitinib dosing and weekly paclitaxel in patients with recurrent EOCs including OC-Pts as well as other solid cancer types (endometrial and primary peritoneal) [65]. Of the 58 patients treated in the study, 3 OC patients showed a partial response, and stable disease was achieved in 10 OC patients. Pharmacokinetic studies showed no significant interactions when linsitinib was administered 2 h prior to paclitaxel. The most common drug-related toxicities were fatigue, nausea, hyperglycemia and drug eruption. Other details of the study outcomes related to PFS have not yet been published.
\nMany compounds are constantly being screened for IGF-IR inhibitory activity, but the similarity between the IGF-IR and the IR receptor presents a challenge for developing selective inhibitors for the IGF-IR. The main concern with this lack of selectivity is that dual inhibitors of IR and IGF-IR, has resulted in hyperglycemia in many clinical trials. This is a major hurdle to overcome in this area of OC therapeutics.
\nThe epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase family of growth factor receptors. These receptors play a direct role in regulating cell proliferation, apoptosis, survival, cell differentiation, and migration [14]. The ERbB family of receptor tyrosine kinases includes EGFR (also known as HER1/ErbB1), EGFR2 (HER2/neu/ERbB2), HER3/ErbB3, and HER4/ErbB4 [66]. Dysregulation of the EGFR function has been linked to the pathology of OC [14] but evidence is conflicting; as other studies have not found strong evidence of a direct link between EGFR expression and function and OC progression. Many factors have been suggested for the mixed results; these include variability in experimental methods, detection procedures, and scoring metrics. Despite the variable study outcomes in OC, evidence supports dysregulated EGFR ligand and receptor expression, heterologous regulation by GPCR ligands, and other non-ligand stimuli initiating chronic activation of EGFRs [14]. This chronic stimulation favors tumor development and progression [14].
\nThe current therapeutic strategy is to inhibit EGFR activity using small molecule tyrosine kinase inhibitors or monoclonal antibodies [67]. Clinical trials have been conducted using the following agents alone and in combination: cetuximab, gefitinib, erlotinib, trastuzumab, and pertuzumab. These treatment regimens were evaluated in patients with recurrent or progressive disease, platinum-sensitive disease, and platinum-resistant/refractory disease among others [67].
\nOf note, the PENELOPE phase III trial investigated the efficacy of pertuzumab in combination with chemotherapy (single-agent topotecan, weekly paclitaxel, or gemcitabine) for treatment of platinum-resistant patients with downregulated human epidermal growth factor 3 (HER3) mRNA expression [68]. The results showed no significant improvement in PFS for the primary analysis (stratified hazard ratio, 0.74; 95% CI, 0.50–1.11; P = 0.14; median PFS, 4.3 months for pertuzumab plus chemotherapy versus 2.6 months for placebo plus chemotherapy). The study concluded that pertuzumab has the potential to be investigated further despite the lack of significance. To date, clinical trials evaluating anti-EGFR and HER therapies have shown minimal improvement in OC-Pt treatment outcome. Further studies evaluating inhibitors of downstream signaling and simultaneous antagonism of the EGFR and HER have been recommended [66].
\nCurrent chemotherapeutic regimens for OC-Pt patients whether monotherapy or combinatorial are inadequate. Immunotherapeutic approaches are now being increasingly explored for these patients where a therapeutic ceiling has been reached with standard chemotherapy. Immunotherapy in OC-Pt patients is just emerging and is currently restricted to clinical trials that have shown promising results. The American Cancer Society defines cancer immunotherapy as ‘treatment that uses your body\'s own immune system to help fight cancer’. Within the tumor microenvironment, the pathological interactions between cancer cells and immune cells is complex and most events spiral into an immunosuppression that causes tumor cells to proliferate and evade immune system attack [69]. There are several categories of immunotherapeutic agents that either stimulate the body’s immune system’s ability to eradicate cancer cells (e.g. cancer vaccines and adoptive T cell transfer), target proteins on the surface of T cells that prevent them from attacking cancer cells (e.g. immune checkpoint inhibitors), or identify specific abnormalities on the surface of cancer cells that render them susceptible to targeted agents (e.g. monoclonal antibodies) [69]. Many of these drugs are being evaluated in OC-Pt patients and are discussed below.
\nCheckpoint proteins are molecules found on the surface of T cells that prevent them from attacking cancer cells [70]. Two such proteins are cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) [71]. PD-1 is expressed on the surface of activated T cells and its ligands, PD-L1 and PD-L2 are found on the surface of dendritic cells or macrophages [70]. Interaction of PD-1 with either PD-L1 or PD-L2 results in inhibition of T cell signaling, reduction in T cell numbers, and increased susceptibility of T cells to apoptosis [71]. CTLA-4 regulates T cell priming and activation in the initiation phase of the immune response [71]. The high expression of PD-L1 and PD-L2 on OC cells is associated with shorter PFS [72]. Similarly, evidence suggests that OC patients with low CTLA-4-mediated signals have a better prognosis than patients with high CTLA-4 activity [73].
\nSeveral antibodies directed against PD-1 (pembrolizumab, nivolumab, and avelumab), PD-L1 (atezolizumab and durvalumab), and CTLA-4 (ipilimumab) have been evaluated in OC. Nivolumab (Opdivo) is a fully humanized IgG4 antibody that blocks the engagement of PD-1-by-PD-1 ligands [74]. Nivolumab was administered every 2 weeks to patients with advanced or relapsed OC-Pt and response rate was assessed by RECIST [74]. The study included 15 OC-Pt patients and the drug showed encouraging clinical efficacy. Some adverse drug reactions including fever, disorientation, and gait disturbance were observed. A dose escalation study (Study ID#: UMIN000005714) is now under way as a second arm of this trial.
\nAvelumab (Bavencio) is a fully human monoclonal antibody of isotype IgG1 that targets PD-L1. It was evaluated in a phase Ib (Study ID#: NCT01772004) expansion study in 75 patients with recurrent/refractory OC which included OC-Pt [75]. Of this cohort, 8 patients showed a partial response and 33 patients displayed stable disease, which was reported as a disease control rate of 54.7%.
\nOne other phase Ib study (KEYNOTE-028/Study ID#: NCT02054806) evaluated the anti-tumor activity and safety of pembrolizumab (Keytruda) in patients with PD-L1 positive advanced OC which included patients refractory to platinum therapy [76]. Pembrolizumab is a humanized antibody that binds to and blocks PD-1. PD-1 blockade with pembrolizumab was well tolerated and displayed anti-tumor activity. Of the 26 patients enrolled in the study, 1 achieved complete response, 2 partial response, and 6 had stable disease. The most common adverse events were fatigue (42.3%), anemia (30.8%), and decreased appetite (30.8%).
\nThe role of the PD-1/PD-L1 axis is continuously been studied and characterized in OC and with new information on OC-Pt immunogenicity emerging consistently, this disease is expected to remain a focused target of PD-1/PD-L1 based therapeutics.
\nInhibition of CTLA-4 during the T cell priming/activation step leads to dysregulated expansion of auto-reactive T cells, including tumor-specific T cells [73]. The anti-CTLA 4 monoclonal antibody ipilimumab (Yervoy) has shown anti-tumor effect in stage IV OC. Ipilimumab is a recombinant human monoclonal antibody (IgG1 kappa immunoglobin) that antagonizes the CTLA-4 immune checkpoint. The administration of ipilimumab to 11 stage IV OC patients previously vaccinated with granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified irradiated autologous tumor cells showed promising results [77]. Ipilimumab caused a reduction or stabilization of CA-125 levels in these patients and no serious toxicities directly attributable to the antibody were observed.
\nTremelimumab is a fully human IgG2 monoclonal antibody to CTLA-4. The combination of tremelimumab with the immunotherapeutic agent durvalumab is currently undergoing a phase I trial (Study ID#: NCT01975831) which includes OC-Pt patients [78]. The primary endpoints of this study are to evaluate safety and identify the maximum tolerated dose of the combination. The secondary objectives are to determine effects on tumor response and PFS. Preliminary data show that the combination has a manageable safety profile, with evidence of clinical activity. Trials with anti-CTLA-4 inhibitors in other cancer types have been associated with significant immune-related toxicities [79], and this might be the major limitation in terms of advancing their application in OC-Pt. More clinical trials are needed in this area of OC-Pt therapeutics.
\nThe aim of vaccinations in cancer patients is to sensitize the immune system to recognize, target, and eradicate tumor cells in an approach that employs both adaptive and innate immunity [80]. Vaccines aim to provoke a tumor-specific immune response by increasing tumor-associated antigen (TAA) presentation by antigen-presenting cells (APCs) which subsequently generates tumor-antigen specific cytotoxic T lymphocytes [80].
\nDendritic cell, peptide, and recombinant viral vaccines are the main types currently undergoing clinical trials for OC. One promising TAA for dendritic cell vaccines is mucin 1 (MUC-1). MUC-1 is a heavily glycosylated, type 1 transmembrane protein that is overexpressed in a large number of cancers including OCs [81]. While multiple MUC-1 vaccines are now in development, CVac (developed by Prima BioMed) is the leading candidate for OC. In the CAN-003 phase II study, 63 confirmed Stage III or IV OC patients received CVac [82]. While the study cohort did not disclose if the patient cohort included OC-Pts, CVac demonstrated positive trends in progression free survival and immune responses and further studies in OC-Pt patients are warranted.
\nA dendritic cell vaccine pulsed with autologous hypochlorous acid-oxidized OC lysate was also evaluated in a pilot study (Study ID#: NCT01132014) of five subjects with recurrent OC [83]. Of the five patients who received the DC vaccine, two had PFS of 24 months or more.
\nPeptide vaccines rely primarily on the immunogenicity of the injected peptides to stimulate an immune response. In the cancer setting, the peptides chosen for the vaccine are TAAs.
\nA phase I trial of the NY-ESO-1 OLP vaccine showed promising results in advanced OC patients that initially received chemotherapy with at least one platinum-based chemotherapy regimen [84]. NY-ESO-1 OLP contains synthetic overlapping long peptides (OLP) from the cancer-testis antigen NY-ESO-1 [84]. The vaccine was found to be safe and rapidly induced consistent integrated immune responses in nearly all vaccinated patients. A phase I/IIb multi-center study was also conducted to evaluate the safety and immunogenicity of the anti-idiotypic antibody vaccine ACA125 in 119 patients with advanced ovarian carcinoma (including OC-Pt patients) [85]. ACA125 functionally imitates the tumor antigen CA125. Preliminary evidence demonstrated safety and immunogenicity of the vaccine. The study data has not reveal conclusions regarding OC-Pt subgroups and this requires further evaluation.
\nRecombinant viral vaccines utilize genetically modified viruses as vectors for introducing TAA-encoding DNA into cells within the body. PANVAC is a vaccine with payload delivered through two viral vectors: recombinant vaccinia and recombinant fowlpox [86]. The vectors contain transgenes for the tumor-associated antigens epithelial mucin 1 (MUC-1) and carcinoembryonic antigen (CEA). Overexpression of MUC-1 and CEA is seen in OC [87, 88]. In a pilot study of PANVAC in 14 OC patients (including OC-Pt), median time to progression was 2 months and median OS was 15.0 months [86].
\nAdoptive cell therapy (ACT) involves the infusion of tumor antigen cells to stimulate innate anti-tumor immunity and induce cancer regression [89]. A pilot study in which seven patients with recurrent local OC were given multiple cycles of intraperitoneal infusions of autologous MUC1 peptide-stimulated cytotoxic T lymphocytes has been completed [90]. Clinical benefit was seen in only one patient who was disease free >12 years. While it is difficult to interpret this information in the context of OC-Pt, the study is worth mentioning as at least one patient had received prior platinum therapy.
\nA phase I clinical trial of adoptive transfer of folate receptor-alpha-redirected autologous T cells for recurrent OC cancer was initiated to establish the safety and proof of concept of autologous FRα-redirected T cells administered intravenously, in subjects with recurrent stage II to IV FRα-positive epithelial ovarian carcinoma (including OC-Pt subgroups) [91]. It is also possible that ACT can be used in combination strategies but the challenge with solid tumors such as OCs; is that tumor microenvironment immunity can cause immunosuppression and render ACT ineffective.
\nToll-like receptors (TLRs) comprise a family of 13 receptors found on hematopoietic and nonhematopoietic cells [92]. The TLR8 subtype is mainly found in monocytes and dendritic cells and it plays an important role in the immune response by recognizing single-stranded RNAs as its natural ligand. Motolimod (Motolid/formerly known as VTX2337) is a synthetic, small molecule, selective agonist of TLR8 that stimulates natural killer cell activity and enhances antibody-dependent cellular cytotoxicity [92]. A phase II randomized, double-blind, placebo-controlled study (Study ID#: NCT01294293), evaluated chemo-immunotherapy combination using motolimod with PLD in recurrent or persistent OC [92]. While the addition of motolimod to PLD did not significantly improve overall survival or PFS, the combination was well tolerated, with no synergistic or unexpected serious toxicity. Another phase II study is also now underway (Study ID#: NCT01666444) in patients with recurrent or persistent epithelial ovarian, fallopian tube, or primary peritoneal cancer. The purpose of this study is to compare the overall survival of patients treated with motolimod + PLD versus those treated with PLD alone in women with recurrent or persistent, epithelial ovarian, fallopian tube, or primary peritoneal cancer. This study will provide further insight on the future of motolimod in OC-Pt.
\nOver the past decade we have learned that OC in general responds poorly (11–25% overall) to single-agent immunotherapy; especially checkpoint blocking strategies [93]. There is very limited data regarding response rates of OC-Pt subgroups specifically, but in most cases these cohorts of patients are integrated in general OC study data, suggesting similar patterns of response. When reviewed collectively, the data suggest that efficient anti-tumor immune response is likely to require combinatorial therapeutic strategies that simultaneously target different stages of tumor escape. Combinations involving immune checkpoint inhibitors, anti-angiogenic agents, and PARP inhibitors are gaining momentum in clinical OC-Pt research and are highlighted below.
\nCurrently, several trials combining PARP and immune checkpoint inhibitors are ongoing [94]. An open-label dose escalation study (Study ID#: NCT02485990) of tremelimumab alone or combined with olaparib for recurrent or persistent OC is currently recruiting participants. This study is aimed at determining what dose of tremelimumab and olaparib is safe and effective in patients with persistent OC including those with OC-Pt.
\nA phase I/II Study (Study ID#: NCT02484404) of durvalumab in combination with olaparib and/or cediranib for advanced solid tumors including OC-Pt is currently recruiting. The aim of the phase I arm is to determine the safety of the combination of durvalumab with olaparib or cediranib. Phase II studies will determine the efficacy of these combination in treating OC.
\nThe TOPACIO trial (Study ID#: NCT02657889) will evaluate niraparib in combination with pembrolizumab in patients with triple-negative breast cancer or OC-Pt. The primary outcome measures are to determine dose-limiting toxicities of combination treatment with niraparib and pembrolizumab and to determine the objective response rate using RECISTv1.1.
\nThe OCTOVA study (Study ID#: NCT03117933), is currently recruiting participants for a randomized phase II trial investigating the efficacy of chemotherapy plus olaparib and cediranib combination therapy in patients with BRCA-mutated OC-Pt. Patients will be randomized to one of three treatment groups: olaparib only, olaparib and cediranib, and the control group paclitaxel. The aim is to compare efficacy and tolerability of the three treatments.
\nA phase II study (Study ID#: NCT02659384) to evaluate the combination of atezolizumab plus bevacizumab and acetylsalicylic acid in recurrent OC-Pt is currently recruiting. The primary aim is to determine PFS at 6 months by RECIST.
\nThe administration of ipilimumab in 11 patients with metastatic ovarian carcinoma after vaccination with irradiated autologous tumor cells engineered to secrete GM-CSF (GVAX), showed promising results [95]. Three patients achieved stable disease as measured by CA-125 levels, and one patient achieved an objective response by radiographic criteria and maintained disease control over 4 years with regular infusions of anti-CTLA-4 antibody.
\nThere are still many hurdles to overcome in the treatment of OC-Pt but some progress has been made in recent years, especially with the development of new immunotherapeutic agents. The good news is that OC cancer is a targetable tumor and although the OC-Pt subgroup of patients have biologically distinct tumors, both targeted therapies and immunotherapy offer an opportunity to uniquely address these differences. As new agents are developed in these categories, the main challenge with existing and future clinical trials will be the risk of adverse events and toxicities, especially with combination immunotherapeutic regimens, where there is an elevated risk for adverse immune events. A second challenge is the optimization of the dose and schedule of immunotherapeutic combinations in order to maximize the overall risk-benefit profile of a given combination. This requires multiple clinical trials with dose escalation studies that can be expensive. This approach is necessary however, especially in the setting of platinum-resistant OC cancer where much research is still needed.
\nThe influenza A M2 protein is a homotetrameric channel [1] that is particularly selective for protons [2] and is essential for uncoating of the virus [3]. The proton selectivity is due to the cluster of His37 imidazole side chains in the channel [4, 5]. This channel has been a primary antiviral target. Amantadine (AMT) and rimantadine (RMT) were highly successful as M2 blockers [6, 7, 8], but they became ineffective in 2005 when a mutation from serine to asparagine at residue 31 (S31N) in M2 occurred [9, 10].
Attempts have been made to develop variants of AMT, RMT and others that could block the V27A, L26F, or S31N mutations [11, 12, 13, 14]. We explored a different approach that could, in theory, target all functional forms of M2 [15].
Drawing from the observation that divalent cations, particularly copper, block M2 current [16] binding in the His37-Trp41 side chain quadruplex [17], divalent copper complexes of AMT were synthesized and found to be effective influenza A inhibitors with reduced cytotoxicity compared to CuCl2 [15]. Because Cu2+ binds strongly to imidazole, it was suggested that the Cu2+ complexes also block M2 through His37-imidazole binding. In addition, the His37 cluster is highly conserved in nature [18], making it a prime target in the M2 channel.
The copper ligands developed were based on AMT and the lesser-known, equally effective M2 WT blocker, cyclooctylamine (CO) [19, 20], and extended via the amine with the functional groups iminodiacetate or iminodiacetamide. Six Cu2+ complexes (Figure 1) were synthesized and characterized using NMR, IR, MS, UV-Vis, and ICP-MS. The complexes demonstrated H37-specific block of M2 current in two electrode voltage-clamp (TEVC) studies with low μM potencies. The copper-free ligands did not show proton current block, demonstrating that the copper was key to the current-blocking process [15].
Copper complexes with the functional groups iminodiacetate or iminodiacetamide extended via the amine to either AMT or CO.
Because of the reduced toxicity to cultured cells found previously, we were interested to learn whether the six metal complexes were toxic to simple organisms. Zebrafish embryos were chosen because they have immune and nervous systems similar in many ways to more advanced organisms, because they are in an early, vulnerable stage of development, and because the compounds are readily administered at infection-relevant concentrations in their bathwater. We also explored and report additional properties of these copper complexes, including their efficacies in the cytopathic effect antiviral assay, their binding to albumin, mutagenicity testing in a bacterial assay, virus resistance development when passaged with cell culture in the presence of the compounds, and molecular dynamics simulations to explore how well the compounds fit in the M2 channel.
Confluent MDCK cells were transferred into 60 wells of a 96-well plate in DMEM (Gibco Thermo Scientific Waltham, MA, 4.5 g/L D-Glucose) with 5% Fetal Bovine Serum (FBS, Hyclone, Logan, UT). The cells were washed with a diluted solution of 50% SEM/50% serumless DMEM. SEM (simple electrolyte medium) consists of 4.33 g NaCl, 0.244 g KCl, 0.103 g CaCl2·2H2O, MgCl2·6H2O, Na2HPO4·7H2O, NaH2PO4·H2O in 500 ml H2O. The cells were incubated for an hour with activated A/WS/33 virus and then the media with virus was removed. The SEM/serumless DMEM with 100 μM metal complex was added to six wells. The complexes were then serial-diluted in two-fold increments six times. Six wells were used as positive controls with no complex or virus added. Six wells were used as negative controls with only virus added and no complex. About 80 μM ribavirin (Sigma-Aldrich, St. Louis, MO) was added to six wells as a positive control. The plates were incubated for 48 h at 33C.
The crystal violet staining technique described previously [21] was used to determine the fraction of cells that survived the exposure to the virus. After 48 h, the test medium was removed, and the cells were washed three times with 150 μl PBS. The cells were stained for 10 min with 50 μl crystal violet solution (0.03% crystal violet (w/v) in 20% methanol). The cells were then washed three times with 150 μl distilled water before adding 100 μl lysis buffer. After 20 min, the optical density (OD) of each well was measured at 590 or 620 nm and averaged over the set of six wells for each concentration.
Because viral dosing was sufficient to eliminate essentially all cells in treatment-free controls, their average OD was subtracted as baseline from the average of the treated well ODs. The result was divided by the average of the uninfected control well ODs to obtain a normalized vitality. Because the vitality can be affected by both reduction of virus cytopathic effect and increase of treatment toxicity as concentration is increased, we fitted the normalized concentration-dependent vitality, V(C), with a joint probability function:
Here, EC50 is the 50% effective dose of treatment that prevents viral cytopathic effect, CC50 is the 50% cytotoxic dose of the treatment, and n1 and n2 are their respective Hill coefficients. If the selectivity index, CC50/EC50, and the Hill coefficients are sufficiently high, this function rises to unity at doses that are sufficient to prevent viral replication but below toxic levels. Non-linear least squares fitting weighted with standard errors of means was done with the Marquardt algorithm in KaleidaGraph4 (Synergy Software, Reading, PA). In practice, it was necessary to fix the Hill coefficients to evaluate the effective doses, then manually adjust the Hill coefficients to improve the fit (due to low numbers of data points). Hence, the reported standard errors of the parameters obtained from the error matrix may be underestimated.
Each copper complex was dissolved in 25 ml of water to obtain a 1 mM and 800 μM solution. All water used in the protein binding assay was collected from a Millipore first-generation beige Milli-Q system. These solutions were sonicated until the crystals were fully dissolved. Four 1:2 serial dilutions were performed from the 800 μM solution to obtain 400, 200, 100, and 50 μM solutions, and a 1:5 dilution was performed from the 50 μM solution to obtain a 10 μM solution. 13.3 mg of BSA was then dissolved in 10 ml of each solution. The solutions were mixed thoroughly and allowed to stand at room temperature for approximately 20 min.
Spin filtration was performed using a swinging bucket rotor at 4000 rpm for 6 min. The spin filters used were Amicon Ultra-15 centrifugal filters. The filtrates from each spin were collected to test for copper content in ICP-MS. Solutions for ICP-MS were prepared from both the original solutions and the filtrates. For each solution, 1 ml of solution was added to 1 ml of 4% HNO3 and 8 ml of 2% HNO3 to obtain a 1:10 dilution of each solution in 2% HNO3. Nitric acid used for ICP-MS analysis was OmniTrace trace-metal grade obtained from EMD Millipore Corporation. We used BSA to model copper binding histidine in solution and calculate relative dissociation constants (Kd) for each complex. Copper concentrations were obtained using ICP-MS. The data was fit to Eq. (2) to estimate Kd and the number of binding sites, n.
Following an approved BYU IACUC protocol, two AB wild-type male and female zebrafish were placed in an embryo media filled tank. The fish remained in a light and temperature-controlled facility until the following morning. Later that day, the fish were transferred into original tank. Embryos were moved into embryo media filled petri dishes (60 embryos/dish) and housed in an incubator for 2 days. Media was changed daily.
Fish embryos were dechorionated at 48 hpf. In a multi-well plate, 10 embryos were selected and 5 were added to each of two wells for each concentration with fresh embryo media. Drug solution (0–200 μM) was then added to test toxicity and observed over 5 days. Drug solutions were changed daily. After 5 days, the fish were scored using a morbidity scale (Table 1) indicating response, spine shape, edema, equilibrium, and death. The average for each complex was normalized using the maximal morbidity score of 50/well. The fish were then euthanized.
Zebrafish scoring indicators | ||||
---|---|---|---|---|
Morbidity points | 0 | 1 | 2 | 3 |
Equilibrium | Upright position | Lying on side | NA | NA |
Response | Quick escape | Sluggish escape | No escape | NA |
Spine shape | Straight | Slightly curved | Strongly curved | NA |
Edema | None | 1 place and minor | 2 places or major | 2 places and major |
Death | =10 |
Scoring indicators observed daily for 5 days.
Five fish per group in each of two wells.
The Modified Ames ISO kit (Environmental Bio-Detection Products Inc., Mississauga, ON) was used with S. typhimurium TA100 (no S9 fraction).
The complexes were compared against the mutagenicity of a positive control (NaN3) and vehicle (water). The complexes were serially diluted 1:2 to compare the complexes’ mutagenic ability at each of six concentrations.
TA100 was hydrated and incubated with histidine overnight at 37°C. Following the kit’s instructions, in 96-well plates’ exposure solution, diluted bacteria mix, and serial two-fold dilutions of complexes were combined with reversion media containing Bromocresol Purple, which serves as a pH indicator to identify infected wells. The 96 well plates were incubated for 6 days at 37°C without agitation. When a sample is mutagenic, it will revert the bacteria to WT, causing the media to turn slightly acidic and show a yellow color.
The number of reverted wells with complex was compared to the average number of reverted wells in the negative control. Significance was calculated using a one-tailed t-test.
The 2KQT M2 structure was used and oriented in a DMPC lipid bilayer with a center-of-mass harmonic constraint. The copper complexes were oriented such that the copper was near (~2.0 Å) at least one of the four imidazole nitrogens. Water molecules within 2.2 Å of the complexes were deleted. The protein-bilayer system was solvated with a tetragonal 60 Å × 60 Å × 90 Å water box as shown in Figure 2. The system was minimized for 1000 steps of steepest descent and heated to 300 K. The M2 channel was equilibrated for 1 ns. The complexes were pulled using a constant force for 10 ps during the production runs. Frames were saved every 50 steps, which is every 50 fs, of production for a total of 200 frames. Standard CHARMM version 37b1 parameters were used. Copper dihedral parameters were created using a 20 kcal/mol/rad2 energy penalty, which kept a conservatively rigid structure throughout the channel (Table A1).
Solvated DMPC-2KQT system.
The distance between imidazole nitrogens and copper on the complexes was calculated using CHARMM’s CORREL subroutine for each frame. The time for each complex was recorded when the copper reached 30 Å away from the imidazole nitrogens. This distance was chosen to represent the complex leaving the mouth of the channel.
The pulling force for each of the complexes was determined by normalizing the pulling forces to a 2.34 nN pulling force on AMT. The 2.34 nN force allowed comparisons to be made between compounds as they left the channel on the 10 ps timescale.
These steered molecular dynamics (SMD) simulations were analyzed by computing the mean and standard deviation of five independent. The five independent simulations were assigned random starting velocities and then analyzed to explore the time needed to pass the 30 Å threshold relative to the starting point from the copper atom on the complex.
The analysis examined whether the pulling forces, copper ligation mechanism, or scaffold (CO, AMT, or neither), significantly affected the exit times relative to free Cu2+.
MDCK cells were seeded into a six-well plate and grown in Dulbecco’s Modified Eagles Medium (DMEM, Sigma-Aldrich, St. Louis, MO) augmented by 5% with fetal bovine serum (FBS, Hyclone, Logan, UT) until confluent. After 48 h, the growth media was removed and replaced with DMEM. At this point the virus (A/CA/07/09) was introduced into the medium (200 pfu/ml) and allowed to adsorb for 1 h. The medium was then removed and replaced with fresh DMEM containing a specified concentration of complex and 5 ml of tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Thermo-Fischer Scientific, Waltham, MA, 1 mg/ml) was added to activate the virus. The plate was incubated at 33°C for 3 days. Then the medium was removed and centrifuged at 2000 rpm in order to remove cell debris. This virus-containing medium was then separated into 1-ml aliquots and frozen in Eppendorf tubes at −80°C. This process was repeated for each successive passage.
The concentration of virus was determined through an immunofluorescence assay (as previously described by [22]), which gave a multiplicity of infection (MOI) of 0.6. MDCK cells were seeded onto glass coverslips in vials containing 1 ml DMEM and trypsin in order to obtain 90% confluency after 24 h. The cells were allowed to grow overnight at 37°C, after which the growth medium was removed and replaced with DMEM. The sample of virus was then diluted by factors of 10, and the various dilutions of virus were stirred into the vials with coverslips. They grew at 33°C for 18 h. After this incubation period, the medium was removed, the cells were fixed with cold acetone (−80°C), and the coverslips were washed and stained with a fluorescein isothiocyanate labeled anti-IAV monoclonal antibody (Millipore Sigma, Burlington, MA, Cat. #5017). Excess antibody was washed off using a solution of 0.05% Tween20 in phosphate buffered saline and then again with distilled water. They were then viewed microscopically and individual infected cells (miniplaques) were counted.
This same process was followed in determining the new EC50 against the specific complex of each resistant strain. Except, 100 pfu of virus was used in each vial. Several different concentrations of the complex with which it was passaged were introduced into the vials, with concentrations ranging from 2 to 70 μM. The cells were infected with the virus in a solution of SEM rather than DMEM. The EC50 was calculated in KaleidaGraph using the Levenberg-Marquardt algorithm. The fitting parameters (sigmoidal function) were used to calculate the EC50 and the standard error of the mean.
To sequence the genome, the viral sample was concentrated 10-fold using a spin filter (VWR North America, Radnor, PA, Cat. #82031-352). After that, viral RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD). The isolated RNA was stored at −20°C. RNA was then reverse-transcribed using Invitrogen’s Superscript III One-Step RT-PCR Platinum Taq HiFi kit (Thermo-Fischer Scientific, Waltham, MA).
The resulting isolated DNA was stored at −20°C. The DNA was then amplified with PCR using the Phusion High-Fidelity PCR kit (New England Biolabs, Ipswich, MA). The solution was purified using Qiagen’s QIAquick PCR Purification Kit (Qiagen, Germantown, MD). It was sequenced using custom forward (TGTAAAACGACGGCCAGTACGAAAAGCAGGTAG) and reverse (CAGGAAACAGCTATGACCAGTAGAAACAAGGTAGT) primers for the segment of the new DNA that codes for the M2 protein.
Although 1–4 had good potency against initial infections in the immunofluorescence (miniplaque) assay [15], the copper complexes had no effect in the cytopathic effect (CPE) assay with MDCK cells when dissolved in serumless DMEM. However, when the serumless DMEM was diluted with SEM, 1 (Figure 3) and to a lesser extent 3 (data not shown) exhibited cell protection. Using a dual-sigmoidal function curve fit, 1 has an EC50 of 0.9 ± 0.08 μM and a CC50 of 5.8 ± 0.37 μM. The submaximal efficacy is due to high cytotoxicity. The selectivity index for 1 is 6.44, given by the ratio of the CC50 and EC50. The low EC50 compares favorably to the EC50 in the miniplaque assay, 6.7 ± 1.2 μM. In contrast, 3 has an EC50 greater than 100 μM, whereas its potency in the miniplaque assay was EC50 = 0.7 ± 0.1 μM, and 2 and 4–6 showed no effect, indicating that other factors were involved. The fact that some efficacy is observed when the medium is diluted with amino-acid free SEM suggests that free amino acids in non-dilute DMEM interfere with the copper complex efficacy.
CPE assay showing protective effect of 1 against A/WS/33 (M2 S31N) infection of MDCK cells using dilute medium (50% DMEM, 50% SEM). MOI 0.6; 48-h incubation.
To illustrate the potential of the metal complexes to bind to proteins, binding to BSA was measured in which a protein solution was mixed with various concentrations of a CuCl2 or copper complex solution. The copper content of the original sample was measured and compared to that of the filtrate. Taking the volume proportions into account, the “free copper concentration” in the filtrate relative to the “total copper concentration” in the original sample was fitted to a model assuming that each protein molecule had n equivalent copper or copper complex binding sites. Table 2 shows the best fit Kd values, assuming that each albumin monomer has n equivalent binding sites. The two parameters interacted and were therefore poorly constrained in the optimization of the deviations squared, but Table 2 indicates that the number of binding sites is well above 10, consistent with the count of 13 surface histidines in monomeric albumin (Figure A1). Complexes 1, 3, and 5 have larger Kd values compared to that of CuCl2 (59.1 μM). This indicates that the ligands on the metal complexes reduce the binding affinity for albumin binding sites, but also still allow for substantial binding. It is also consistent with the electrophysiology results for blocking through binding of copper complex to the His37 cluster in the M2 channel.
Complex | Kd (μM) | Sites (n) |
---|---|---|
CuCl2 | 59.1 | 15 |
1 | 128.7 | 19 |
3 | 179.5 | 20 |
5 | 380 | 20* |
Binding to bovine serum albumin results.
The value of n was not well-constrained and was therefore fixed during the curve fit.
Kd values for representative compounds 1, 3, and 5.
BSA has 13 surface histidines (Figure A1), however, all of the fits optimized n at >13 copper binding sites. This difference could suggest non-specific binding to other sites on BSA. The high Kd’s for the complexes relative to CuCl2 indicate that the complexes remain intact during binding to BSA. The binding the copper complexes to BSA is very weak compared to that of the M2S31N (AMT resistant) channel, where block was ~80% for 1 and 3 after 57 and 27 min perfusion, respectively. This suggests that protein binding in vivo would be a minor concern. However, it is clear that binding by non-M2 proteins is detectable and, given their large quantity inside and outside the blood, they could limit access of the copper complexes to virus.
Toxicity was evaluated for zebrafish exposed to various concentrations of CuCl2 or copper complex (1–5) added as methanolic solutions to the embryo bath medium starting 48 h post fertilization (Day 0) (Figure 4). At 200 μM copper complex on day 1, compounds 2, 4, and 5 show minimum toxicity effects, 1 and 3 show moderate toxicity including slow response to stimulation, slightly curved spine, and minor edema, whereas CuCl2 causes major edema, strongly curved spine, no response to stimulation, and death. By day 2 at 200 μM, the toxicities of 1, 2, 4, and 5 have increased moderately but still only moderate spine curvature and minor edema, while 3 causes slow response to stimulation, strongly curved spine, and moderate to major edema. By days 3, 4, and 5 at 200 μM, all but 5 show low or no response, strongly curved spines, major edema, and some death. The MeOH vehicle controls showed statistically insignificant toxicity.
Zebrafish toxicity of copper complexes. CuCl2 (yellow), 1 (red), 2 (blue), 3 (green), 4 (magenta), 5 (black). (Arbitrary toxicity index, see Section 2).
Compared to CuCl2, the copper complexes show less toxicity, suggesting that the ligands are coordinating to the copper and helping to reduce its toxicity through day 2 of high dosage. All of the copper complexes produce some toxicity in the zebra fish for all experimental concentrations, but compound 5 does not increase in toxicity over time as much as the other complexes. This suggests further testing and modification of compound 5 could lead to a safe anti-influenza A therapeutic.
The mutagenicity of the copper complexes was tested using the Ames test. Table 3 shows the percent reversion out of 48 wells of three complexes. They were tested for mutagenicity against S. Typhimurium TA100, which strain of bacteria allows a test for mutagenicity caused by base-pair substitution and oxidative damage. The percent of revertant wells (reversion rate) was compared against the negative control and found to be statistically insignificant (p > 0.01). The positive control (NaN3) had an average 91.7% reversion rate compared to the negative control’s average rate of 43.8% (p < 0.0001). Complexes 1, 3, and 4 did not show significant rate of reversion at any tested concentration compared to NaN3. The copper complexes showed approximately the same reversion rates as the negative control after 6 days. Therefore, they do not cause mutagenicity due to base-pair substitution or oxidative damage.
Concentration (μM) | Complex | ||
---|---|---|---|
1 | 3 | 4 | |
500 | 42 | 42 | 50 |
250 | 42 | 42 | 30 |
125 | 31 | 52 | 33 |
62.5 | 31 | 41 | 38 |
31.25 | 52 | 58 | 67 |
15.63 | 56 | 52 | 56 |
0 | 35 | 46 | 50 |
Ames mutagenicity assay results.
Percent of reversion out of 48 wells for compounds 1, 3, and 4 for concentrations between 15 and 500 μM. 0 is water with no complex.
Because the putative target for the metal complexes, the His37 quadruplex, is highly conserved in nature and functionally critical for vRNP uncoating, we explored the propensity for virus resistance formation with passaging in MDCK cell cultures. Because the incubation had to be done in DMEM, which is known to inhibit complex efficacy, we used higher concentrations of complexes for the incubations such that the efficacy of block was projected to be ~50%, thus creating a concentration where mutation could occur. Ten passages (~5 weeks) of incubated virus in DMEM dosed with increasing metal complex concentrations (ranging from 50 to 100 μM) was chosen as a rigorous test. Resistance would be identifiable by an increase in miniplaque EC50 after passaging relative to the original value. As shown in Table 4, the new EC50 (column 3) is comparable to the original EC50 (column 2). Because none of the copper complexes significantly increased the EC50 after 5 weeks of incubation, we conclude that resistance is slow to develop. This contrasts with rapid resistance development when passaging with AMT [15].
Complex | Original A/CA/09 (μM) | 10 passages with complex (μM) |
---|---|---|
1 | 6.9 ± 1.2 | 3.7 ± 0.5 |
2 | 4.9 ± 0.8 | 2.1 ± 1.1 |
3 | 0.7 ± 0.1 | 1.1 ± 0.4 |
4 | 11.6 ± 1.1 | 3.9 ± 6.8 |
5 | 8.2 ± 2.0 | 1.3 ± 0.2 |
6 | 4.4 ± 0.6 | 2.9 ± 0.3 |
Miniplaque EC50 ± SE (EC50) before (original) and after 10 passages of virus in MDCK cells.
DMEM was used for passage incubations and SEM for the miniplaque assays.
The vRNA M segment was extracted from the passaged virus exposed to 3, sequenced and compared to A/CA/07/2009 using a reverse-BLAST mechanism. The only base mutation discovered was G749A, which translates to the amino acid mutation G16E. This amino acid is positioned in the region of the channel entry that is outside of the membrane and unlikely to influence channel permeation. According to the results in the above table, this mutation did not confer resistance to this compound. We consider the occasionally observed natural M2 mutant G34E to be likely to escape block by these complexes. Although we did not see resistance develop in our assays, a more direct assessment of the G34E site mutation using electrophysiology might be instructive about resistance potential for these compounds in future studies.
Constant force steered molecular dynamics (MD) simulations were carried out to explore the steric limitations on metal complex exit from the M2 transmembrane domain AMT binding site. A 2.34 nN force was used to pull the complexes pass the 30 Å threshold and beyond the Val27 cluster within 10 ps. The 2.34 nN force gave a sufficient spread in leaving times to allow assessment of the ease of unbinding relative to AMT. For these simulations, the force was applied to the center-of-mass of the complex. Example trajectories for AMT (green) and 4 (yellow) are shown in Figure 5. The starting configurations (left) had the adamantyl groups of AMT and 4 superimposed with the copper atom of 4 oriented down, close to H37. This binding configuration was used for all of the metal complexes. V27 and H37 are shown as reference points along the channel.
Exit trajectories of AMT and 4 leaving M2 channel. Three of the four M2 TMD monomers from the simulation of 4 are displayed for reference. Valine 27 and Histidine 37 side chains are shown in grey, 4 is yellow and AMT is green. Both complexes are experiencing the same 2.34 nN pulling force.
Table 5 shows the average time to leave from five independent simulations (identical starting configurations, but randomly assigned atomic velocities) for each complex to pass the 30 Å threshold. All metal complexes took longer to leave the channel than AMT. AMT exited the channel in 2.77 ps. Complex 4 interacts with the V27 side chain and was the slowest compound to leave the channel, with its leaving time at 7.05 ps. By 4.4 and 6.75 ps, some distortion is seen on the protein subunit as 4 is pulled further out of the channel.
Complex | Average time to leave (ps) |
1 | 4.60 ± 1.14 |
2 | 6.85 ± 1.01 |
3 | 4.67 ± 1.07 |
4 | 7.05 ± 0.53 |
5 | 3.75 ± 0.89 |
6 | 4.00 ± 1.06 |
AMT | 2.77 ± 0.26 |
Average time to leave (±standard deviation) the channel with a 2.34 nN force applied to the compound’s center-of-mass.
The copper complexes are relatively non-toxic in zebrafish embryos compared to CuCl2 over a 5-day period. Also, they are efficacious in a 3-day assay (but with limitations due to serum protein binding and amino acid interference), are non-mutagenic compared to sodium azide, are slower to leave the M2 binding site compared to AMT, and, also compared to AMT, are not prone to resistance development. In vivo they would face competition with binding to other proteins and the therapeutic window is small. However, complexation of copper could be pharmacologically beneficial.
Further testing of these copper complexes should include isothermal titration calorimetry (ITC) experiments with influenza A M2 channel to obtain binding energies, two-electrode voltage clamp (TEVC) experiments to obtain rate constants of binding to M2, and testing in an animal model that more accurately represents the effects of the copper complexes on humans.
Bovine serum albumin view (RCSB 3V03). Histidine side chains are colored yellow.
CU parameters | |||
---|---|---|---|
Bond | Kb(kcal/mol/Å2) | B0(Å) | |
CU-N | 270.2 | 2.026 | |
Angle | Kθ(kcal/mole/rad2) | θ0(degrees) | |
CT1-NPH-CU | 96.150 | 128.05 | |
HB1-NPH-CU | 30 | 123 | |
CT2-NH3-CU | 96.150 | 128.05 | |
H-NPH-CU | 0 | 180 | |
NPH-CU-NH3 | 14.39 | 90 | |
NPH-CU-NPH | 14.39 | 90 | |
Dihedral | Φ (kcal/mole/rad2) | Multiplicity | Delta (degrees) |
NPH-CU-NH3-CT2 | 20 | 1 | 92.30 |
CU-NPH-C2-OB | 20 | 2 | 169.1 |
CU-NH3-CT1-CT2 | 20 | 1 | −58.6 |
H-NPH-CU-NH3 | 20 | 2 | 157.9 |
NPH-CU-NPH-H | 20 | 2 | 161.2 |
H-NPH-CU-NH3 | 20 | 2 | 157.9 |
C2-NPH-CU-NPH | 20 | 2 | 18.8 |
CU-NPH-C2-CT2 | 20 | 2 | 8.0 |
CU-NH3-CT2-HA | 20 | 3 | 90.20 |
C2-CT2-NH3-CU | 20 | 2 | −30.80 |
NPH-CU-NH3-H | 20 | 2 | 161.2 |
CHARMM copper parameters.
Ove Odredbe i uvjeti ističu pravila i regulacije u svezi korištenja IntechOpenove stranice www.intechopen.com i svih poddomena u vlasništvu IntechOpena, tvrtke sa sjedištem u 5 Princes Gate Court, London, SW7 2QJ, Ujedinjeno Kraljevstvo.
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\\n\\nSljedeća terminologija odnosi se na Odredbe i uvjete, te na sve naše ugovore:
\\n\\nKlijent, stranka, vi, vaš odnosi se na vas, osobu koja pristupa ovoj stranici i prihvaća IntechOpenove Odredbe i uvjete;
\\n\\nKompanija, tvrtka, mi, naše odnosi se na tvrtku IntechOpen;
\\n\\nStranke, strane odnosi se na klijenta i na nas, ili samo na klijenta ili nas.
\\n\\nSve odredbe koje se odnose na ponudu, prihvat ili razmatranje plaćanja, a za koja mi pružamo asistenciju klijentu, bilo na ugovoreni ili fiksni način, a s ciljem da se ostvare potrebe i želje klijenta u svezi s našim uslugama, su podložne zakonskim odredbama Ujedinjenog Kraljevstva.
\\n\\nOsim ako nije suprotno navedeno, IntechOpen i/ili svi davatelji licence vlasnici su intelektualnog vlasništva nad svim materijalima na www.intechopen.com. Sva prava intelektualnog vlasništva su pridržana. Stranice sa www.intechopen.com možete gledati, preuzimati, dijeliti, dijeliti poveznice i printati za osobnu uporabu, a temeljem pravila sadržanih u ovim Odredbama i uvjetima.
\\n\\nMi koristimo kolačiće. Korištenjem IntechOpenove stranice slažete se s korištenjem kolačića u skladu s IntechOpenovom Politikom privatnosti. Većina modernih, interaktivnih stranica koristi kolačiće kako bi omogućila ponovno pronalaženje korisničkih detalja kod svakog posjeta. Na našoj stranici kolačići se uglavnom koriste kako bi omogućili funkcionalnost i olakšali posjetiteljima korištenje stranice.
\\n\\nIntechOpen ili njegovi suradnici niti u jednom slučaju neće biti odgovorni za štete (štete uključuju gubitak podataka ili profita, druge poslovne prekide, te sve ostale štete) koje nastanu zbog korištenja materijala na IntechOpenovoj stranici ili nemogućnosti da se iste koriste, čak i ako je IntechOpen ili njegov predstavnik o takvoj šteti obaviješten pismenim ili usmenim putem. Neke jurisdikcije ne dozvoljavaju ograničenja garancija ili ograničenja obveza za posljedične ili slučajne štete pa se u tom slučaju ova ograničenja možda ne odnose na vas.
\\n\\nMaterijali koji se pojavljuju na IntechOpenovoj stranici mogu sadržavati manje greške, tipfelere ili fotografske greške. IntechOpen može napraviti promjene na bilo kojem materijalu koji se nalazi na stranici u bilo koje vrijeme.
\\n\\nIntechOpen nije formalno povezan niti s jednom vanjskom stranicom čije poveznice vode na www.intechopen.com, osim ako to nije izravno navedeno. Iz tog razloga IntechOpen nije odgovoran za sadržaj koji se pojavljuje na takvim stranicama. Poveznica na IntechOpenovu stranicu ne implicira povezanost sa IntechOpenom. Korištenje takvih poveznica isključiva je odgovornost korisnika.
\\n\\nZadržavamo pravo vlasništva nad cjelokupnom stranicom www.intechopen.com i nad svim materijalom na toj stranici. Koristeći se našim uslugama, slažete se da maknete sve poveznice na našu stranicu odmah nakon što to od vas zatražimo. Također, zadržavamo pravo da ove Odredbe i uvjete, i politiku o poveznicama izmjenimo u bilo koje vrijeme. Koristeći se poveznicama na naše stranice slažete se s ovim Odredbama i uvjetima.
\\n\\nAko smatrate da je bilo koja poveznica na našoj stranici sumnjiva iz bilo kojeg razloga, molimo vas da nas kontaktirate. U tom slučaju razmotrit ćemo micanje poveznice s naše stranice, iako nismo obvezni to napraviti.
\\n\\nBez prethodne privole i izričite pisane dozvole, ne možete stvarati okvire oko naših stranica ili koristiti druge tehnike koje na bilo koji način mogu promijeniti prezentaciju ili izgled naše stranice.
\\n\\nIntechOpen može ove Odredbe izmijeniti u bilo koje vrijeme i bez prethodne obavijesti. Koristeći ovu stranicu vi se slažete s trenutnim Odredbama i uvjetima koje su na snazi.
\\n\\nOve Odredbe i uvjeti su sastavljeni u skladu s odredbama prava Ujedinjenog Kraljevstva, a za sve sporove nadležan je sud u Londonu, Ujedinjeno Kraljevstvo.
\\n"}]'},components:[{type:"htmlEditorComponent",content:"Pristupom na stranicu www.intechopen.com slažete se s ovim odredbama, sa svim primjenjivim zakonskim odredbama, te se slažete s poštovanjem svih lokalnih zakona. Korištenje i/ili pristup ovoj stranici temelji se na potpunom prihvaćanju ovih odredbi. Svi materijali na ovoj stranici zaštićeni su primjenjivim zakonima o autorskim pravima i žigu.
\n\nSljedeća terminologija odnosi se na Odredbe i uvjete, te na sve naše ugovore:
\n\nKlijent, stranka, vi, vaš odnosi se na vas, osobu koja pristupa ovoj stranici i prihvaća IntechOpenove Odredbe i uvjete;
\n\nKompanija, tvrtka, mi, naše odnosi se na tvrtku IntechOpen;
\n\nStranke, strane odnosi se na klijenta i na nas, ili samo na klijenta ili nas.
\n\nSve odredbe koje se odnose na ponudu, prihvat ili razmatranje plaćanja, a za koja mi pružamo asistenciju klijentu, bilo na ugovoreni ili fiksni način, a s ciljem da se ostvare potrebe i želje klijenta u svezi s našim uslugama, su podložne zakonskim odredbama Ujedinjenog Kraljevstva.
\n\nOsim ako nije suprotno navedeno, IntechOpen i/ili svi davatelji licence vlasnici su intelektualnog vlasništva nad svim materijalima na www.intechopen.com. Sva prava intelektualnog vlasništva su pridržana. Stranice sa www.intechopen.com možete gledati, preuzimati, dijeliti, dijeliti poveznice i printati za osobnu uporabu, a temeljem pravila sadržanih u ovim Odredbama i uvjetima.
\n\nMi koristimo kolačiće. Korištenjem IntechOpenove stranice slažete se s korištenjem kolačića u skladu s IntechOpenovom Politikom privatnosti. Većina modernih, interaktivnih stranica koristi kolačiće kako bi omogućila ponovno pronalaženje korisničkih detalja kod svakog posjeta. Na našoj stranici kolačići se uglavnom koriste kako bi omogućili funkcionalnost i olakšali posjetiteljima korištenje stranice.
\n\nIntechOpen ili njegovi suradnici niti u jednom slučaju neće biti odgovorni za štete (štete uključuju gubitak podataka ili profita, druge poslovne prekide, te sve ostale štete) koje nastanu zbog korištenja materijala na IntechOpenovoj stranici ili nemogućnosti da se iste koriste, čak i ako je IntechOpen ili njegov predstavnik o takvoj šteti obaviješten pismenim ili usmenim putem. Neke jurisdikcije ne dozvoljavaju ograničenja garancija ili ograničenja obveza za posljedične ili slučajne štete pa se u tom slučaju ova ograničenja možda ne odnose na vas.
\n\nMaterijali koji se pojavljuju na IntechOpenovoj stranici mogu sadržavati manje greške, tipfelere ili fotografske greške. IntechOpen može napraviti promjene na bilo kojem materijalu koji se nalazi na stranici u bilo koje vrijeme.
\n\nIntechOpen nije formalno povezan niti s jednom vanjskom stranicom čije poveznice vode na www.intechopen.com, osim ako to nije izravno navedeno. Iz tog razloga IntechOpen nije odgovoran za sadržaj koji se pojavljuje na takvim stranicama. Poveznica na IntechOpenovu stranicu ne implicira povezanost sa IntechOpenom. Korištenje takvih poveznica isključiva je odgovornost korisnika.
\n\nZadržavamo pravo vlasništva nad cjelokupnom stranicom www.intechopen.com i nad svim materijalom na toj stranici. Koristeći se našim uslugama, slažete se da maknete sve poveznice na našu stranicu odmah nakon što to od vas zatražimo. Također, zadržavamo pravo da ove Odredbe i uvjete, i politiku o poveznicama izmjenimo u bilo koje vrijeme. Koristeći se poveznicama na naše stranice slažete se s ovim Odredbama i uvjetima.
\n\nAko smatrate da je bilo koja poveznica na našoj stranici sumnjiva iz bilo kojeg razloga, molimo vas da nas kontaktirate. U tom slučaju razmotrit ćemo micanje poveznice s naše stranice, iako nismo obvezni to napraviti.
\n\nBez prethodne privole i izričite pisane dozvole, ne možete stvarati okvire oko naših stranica ili koristiti druge tehnike koje na bilo koji način mogu promijeniti prezentaciju ili izgled naše stranice.
\n\nIntechOpen može ove Odredbe izmijeniti u bilo koje vrijeme i bez prethodne obavijesti. Koristeći ovu stranicu vi se slažete s trenutnim Odredbama i uvjetima koje su na snazi.
\n\nOve Odredbe i uvjeti su sastavljeni u skladu s odredbama prava Ujedinjenog Kraljevstva, a za sve sporove nadležan je sud u Londonu, Ujedinjeno Kraljevstvo.
\n"}]},successStories:{items:[]},authorsAndEditors:{filterParams:{sort:"featured,name"},profiles:[{id:"6700",title:"Dr.",name:"Abbass A.",middleName:null,surname:"Hashim",slug:"abbass-a.-hashim",fullName:"Abbass A. Hashim",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/6700/images/1864_n.jpg",biography:"Currently I am carrying out research in several areas of interest, mainly covering work on chemical and bio-sensors, semiconductor thin film device fabrication and characterisation.\nAt the moment I have very strong interest in radiation environmental pollution and bacteriology treatment. The teams of researchers are working very hard to bring novel results in this field. I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. I have served as the editor for many books, been a member of the editorial board in science journals, have published many papers and hold many patents.",institutionString:null,institution:{name:"Sheffield Hallam University",country:{name:"United Kingdom"}}},{id:"54525",title:"Prof.",name:"Abdul Latif",middleName:null,surname:"Ahmad",slug:"abdul-latif-ahmad",fullName:"Abdul Latif Ahmad",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"20567",title:"Prof.",name:"Ado",middleName:null,surname:"Jorio",slug:"ado-jorio",fullName:"Ado Jorio",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Universidade Federal de Minas Gerais",country:{name:"Brazil"}}},{id:"47940",title:"Dr.",name:"Alberto",middleName:null,surname:"Mantovani",slug:"alberto-mantovani",fullName:"Alberto Mantovani",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"12392",title:"Mr.",name:"Alex",middleName:null,surname:"Lazinica",slug:"alex-lazinica",fullName:"Alex Lazinica",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/12392/images/7282_n.png",biography:"Alex Lazinica is the founder and CEO of IntechOpen. After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\r\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. He is an expert in structural, absorptive, catalytic and photocatalytic properties, in structural organization and dynamic features of ionic liquids, in magnetic interactions between paramagnetic centers. The author or co-author of 3 books, over 200 articles and reviews in scientific journals and books. He is an actual member of the International EPR/ESR Society, European Society on Quantum Solar Energy Conversion, Moscow House of Scientists, of the Board of Moscow Physical Society.",institutionString:null,institution:{name:"Semenov Institute of Chemical Physics",country:{name:"Russia"}}},{id:"62389",title:"PhD.",name:"Ali Demir",middleName:null,surname:"Sezer",slug:"ali-demir-sezer",fullName:"Ali Demir Sezer",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62389/images/3413_n.jpg",biography:"Dr. Ali Demir Sezer has a Ph.D. from Pharmaceutical Biotechnology at the Faculty of Pharmacy, University of Marmara (Turkey). He is the member of many Pharmaceutical Associations and acts as a reviewer of scientific journals and European projects under different research areas such as: drug delivery systems, nanotechnology and pharmaceutical biotechnology. Dr. Sezer is the author of many scientific publications in peer-reviewed journals and poster communications. Focus of his research activity is drug delivery, physico-chemical characterization and biological evaluation of biopolymers micro and nanoparticles as modified drug delivery system, and colloidal drug carriers (liposomes, nanoparticles etc.).",institutionString:null,institution:{name:"Marmara University",country:{name:"Turkey"}}},{id:"61051",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"100762",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"St David's Medical Center",country:{name:"United States of America"}}},{id:"107416",title:"Dr.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Texas Cardiac Arrhythmia",country:{name:"United States of America"}}},{id:"64434",title:"Dr.",name:"Angkoon",middleName:null,surname:"Phinyomark",slug:"angkoon-phinyomark",fullName:"Angkoon Phinyomark",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/64434/images/2619_n.jpg",biography:"My name is Angkoon Phinyomark. I received a B.Eng. degree in Computer Engineering with First Class Honors in 2008 from Prince of Songkla University, Songkhla, Thailand, where I received a Ph.D. degree in Electrical Engineering. My research interests are primarily in the area of biomedical signal processing and classification notably EMG (electromyography signal), EOG (electrooculography signal), and EEG (electroencephalography signal), image analysis notably breast cancer analysis and optical coherence tomography, and rehabilitation engineering. I became a student member of IEEE in 2008. During October 2011-March 2012, I had worked at School of Computer Science and Electronic Engineering, University of Essex, Colchester, Essex, United Kingdom. In addition, during a B.Eng. I had been a visiting research student at Faculty of Computer Science, University of Murcia, Murcia, Spain for three months.\n\nI have published over 40 papers during 5 years in refereed journals, books, and conference proceedings in the areas of electro-physiological signals processing and classification, notably EMG and EOG signals, fractal analysis, wavelet analysis, texture analysis, feature extraction and machine learning algorithms, and assistive and rehabilitative devices. I have several computer programming language certificates, i.e. Sun Certified Programmer for the Java 2 Platform 1.4 (SCJP), Microsoft Certified Professional Developer, Web Developer (MCPD), Microsoft Certified Technology Specialist, .NET Framework 2.0 Web (MCTS). 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