Madhur D. Shastri

Endoplasmic Reticulum Stress and Oxidative Stress: A Vicious Nexus Implicated in Bowel Disease Pathophysiology

The endoplasmic reticulum (ER) is a complex protein folding and trafficking organelle. Alteration and discrepancy in the endoplasmic reticulum environment can affect the protein folding process and hence, can result in the production of misfolded proteins. The accumulation of misfolded proteins causes cellular damage and elicits endoplasmic reticulum stress. Under such stress conditions, cells exhibit reduced functional synthesis, and will undergo apoptosis if the stress is prolonged. To resolve the ER stress, cells trigger an intrinsic mechanism called an unfolded protein response (UPR). UPR is an adaptive signaling process that triggers multiple pathways through the endoplasmic reticulum transmembrane transducers, to reduce and remove misfolded proteins and improve the protein folding mechanism, in order to improve and maintain endoplasmic reticulum homeostasis. An increasing number of studies support the view that oxidative stress has a strong connection with ER stress. During the protein folding process, reactive oxygen species are produced as by-products, leading to impaired reduction-oxidation (redox) balance conferring oxidative stress. As the protein folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and enhance the production of misfolded proteins, causing further ER stress. It is proposed that endoplasmic reticulum stress and oxidative stress together play significant roles in the pathophysiology of bowel diseases.

Non-anticoagulant derivatives of heparin for the management of asthma: distant dream or close reality?

Introduction: Approximately 300 million people worldwide are currently affected by asthma. Improvements in the understanding of the mechanisms involved in such inflammatory airway disorders has led to the recognition of new therapeutic approaches. Heparin, a widely used anticoagulant, has been shown to be beneficial in the management of asthma. It belongs to the family of highly sulphated polysaccharides referred to as glycosaminoglycans, containing a heterogeneous mixture of both anticoagulant and non-anticoagulant polysaccharides. Experimental findings have suggested that heparin has potential anti-asthmatic properties owing to the ability of its non-anticoagulant oligosaccharides to bind and modulate the activity of a wide range of biological molecules involved in the inflammatory process. Areas covered: This review focuses on the potential mechanisms of action and clinical application of heparin as an anti-inflammatory agent for the management of asthma. Expert opinion: Heparin may play a significant role in the management of asthma. However, these properties are often hindered by the presence of anticoagulant oligosaccharides, which possess a significant risk of bleeding. Therefore, its therapeutic potential must be explored using well-designed clinical studies that focus on identifying and isolating the anti-inflammatory oligosaccharides of heparin and further elucidating the structure and mechanisms of actions of these non-anticoagulant oligosaccharides.

Non-Anticoagulant Fractions of Enoxaparin Suppress Inflammatory Cytokine Release from Peripheral Blood Mononuclear Cells of Allergic Asthmatic Individuals

Background Enoxaparin, a low-molecular-weight heparin, is known to possess anti-inflammatory properties. However, its clinical exploitation as an anti-inflammatory agent is hampered by its anticoagulant effect and the associated risk of bleeding. Objective The aim of the current study was to examine the ability of non-anticoagulant fractions of enoxaparin to inhibit the release of key inflammatory cytokines in primed peripheral blood mononuclear cells derived from allergic mild asthmatics. Methods Peripheral blood mononuclear cells from allergic asthmatics were activated with phytohaemag glutinin (PHA), concanavalin-A (ConA) or phorbol 12-myristate 13-acetate (PMA) in the presence or absence of enoxaparin fractions before cytokine levels were quantified using specific cytokine bead arrays. Together with nuclear magnetic resonance analysis,time-dependent and target-specific effects of enoxaparin fractions were used to elucidate structural determinants for their anti-inflammatory effect and gain mechanistic insights into their anti-inflammatory activity. Results Two non-anticoagulant fractions of enoxaparin were identified that significantly inhibited T-cell activation. A disaccharide fraction of enoxaparin inhibited the release of IL-4, IL-5, IL-13 and TNF-α by more than 57% while a tetrasaccharide fraction was found to inhibit the release of tested cytokines by more than 68%. Our data suggest that the observed response is likely to be due to an interaction of 6-O-sulfated tetrasaccharide with cellular receptor(s). Conclusion and Clinical Relevance The two identified anti-inflammatory fractions lacked anticoagulant activity and are therefore not associated with risk of bleeding. The findings highlight the potential therapeutic use of enoxaparin-derived fractions, in particular tetrasaccharide, in patients with chronic inflammatory disorders.

Opposing Effects of Low Molecular Weight Heparins on the Release of Inflammatory Cytokines from Peripheral Blood Mononuclear Cells of Asthmatics

Background T-cell-mediated inflammatory cytokines, such as interleukin (IL)-4, IL-5, IL-13 and tumor necrosis factor-alpha (TNF-α), play an important role in the initiation and progression of inflammatory airways diseases. Low-molecular-weight heparins (LMWHs), widely used anticoagulants, possess anti-inflammatory properties making them potential treatment options for inflammatory diseases, including asthma. In the current study, we investigated the modulating effects of two LMWHs (enoxaparin and dalteparin) on the release of cytokines from stimulated peripheral blood mononuclear cells (PBMCs) of asthmatic subjects to identify the specific components responsible for the effects. Methods PBMCs from asthmatic subjects (consist of ~75% of T-cells) were isolated from blood taken from ten asthmatic subjects. The PBMCs were pre-treated in the presence or absence of different concentrations of LMWHs, and were then stimulated by phytohaemagglutinin for the release of IL-4, IL-5, IL-13 and TNF-α. LMWHs were completely or selectively desulfated and their anticoagulant effect, as well as the ability to modulate cytokine release, was determined. LMWHs were chromatographically fractionated and each fraction was tested for molecular weight determination along with an assessment of anticoagulant potency and effect on cytokine release. Results Enoxaparin inhibited cytokine release by more than 48%, whereas dalteparin increased their release by more than 25%. The observed anti-inflammatory effects of enoxaparin were independent of their anticoagulant activities. Smaller fractions, in particular dp4 (four saccharide units), were responsible for the inhibitory effect of enoxaparin. Whereas, the larger fractions, in particular dp22 (twenty two saccharide units), were associated with the stimulatory effect of dalteparin. Conclusion Enoxaparin and dalteparin demonstrated opposing effects on inflammatory markers. These observed effects could be due to the presence of structurally different components in the two LMWHs arising from different methods of depolymerisation. This study provides a platform for further studies investigating the usefulness of enoxaparin in various inflammatory diseases.

In-Vitro Suppression of IL-6 and IL-8 Release from Human Pulmonary Epithelial Cells by Non-Anticoagulant Fraction of Enoxaparin

Background Enoxaparin, a mixture of anticoagulant and non-anticoagulant fractions, is widely used as an anticoagulant agent. However, it is also reported to possess anti-inflammatory properties. Our study indicated that enoxaparin inhibits the release of IL-6 and IL-8 from A549 pulmonary epithelial cells. Their release causes extensive lung tissue damage. The use of enoxaparin as an anti-inflammatory agent is hampered due to the risk of bleeding associated with its anticoagulant fractions. Therefore, we aimed to identify the fraction responsible for the observed anti-inflammatory effect of enoxaparin and to determine the relationship between its structure and biological activities. Methods A549 pulmonary epithelial cells were pre-treated in the presence of enoxaparin and its fractions. The levels of IL-6 and IL-8 released from the trypsin-stimulated cells were measured by ELISA. The anticoagulant activity of the fraction responsible for the effect of enoxaparin was determined using an anti-factor-Xa assay. The fraction was structurally characterised using nuclear magnetic resonance. The fraction was 2-O, 6-O or N-desulfated to determine the position of sulfate groups required for the inhibition of interleukins. High-performance size-exclusion chromatography was performed to rule out that the observed effect was due to the interaction between the fraction and trypsin or interleukins. Results Enoxaparin (60μg/mL) inhibited the release of IL-6 and IL-8 by >30%. The fraction responsible for this effect of enoxaparin was found to be a disaccharide composed of α-L-iduronic-acid and α-D-glucosamine-6-sulfate. It (15μg/mL) inhibited the release of interleukins by >70%. The 6-O sulphate groups were responsible for its anti-inflammatory effect. The fraction did not bind to trypsin or interleukins, suggesting the effect was not due to an artefact of the experimental model. Conclusion The identified disaccharide has no anticoagulant activity and therefore eliminates the risk of bleeding associated with enoxaparin. Future in-vivo studies should be designed to validate findings of the current study.

The Effect of Undaria pinnatifida Fucoidan on the Pharmacokinetics of Letrozole and Tamoxifen in Patients With Breast Cancer

Background: Although the use of complementary and alternative medicines is widespread in cancer patients, clinical evidence of their benefits is sparse. Furthermore, while they are often assumed to be safe with regard to concurrent use of anticancer therapies, few studies have been carried out to investigate possible interactions. Fucoidans are a group of sulfated carbohydrates, derived from marine brown algae, which have long been used as dietary supplements due to their reported medicinal properties, including anticancer activity. The aim of this study was to investigate the effect of co-administration of fucoidan, derived from Undaria pinnatifida, on the pharmacokinetics of 2 commonly used hormonal therapies, letrozole and tamoxifen, in patients with breast cancer. Methods: This was an open label non-crossover study in patients with active malignancy taking letrozole or tamoxifen (n = 10 for each group). Patients took oral fucoidan, given in the form of Maritech extract, for a 3-week period (500 mg twice daily). Trough plasma concentrations of letrozole, tamoxifen, 4-hydroxytamoxifen, and endoxifen were measured using HPLC-CAD (high-performance liquid chromatography charged aerosol detector), at baseline and after concomitant administration with fucoidan. Results: No significant changes in steady-state plasma concentrations of letrozole, tamoxifen, or tamoxifen metabolites were detected after co-administration with fucoidan. In addition, no adverse effects of fucoidan were reported, and toxicity monitoring showed no significant differences in all parameters measured over the study period. Conclusions: Administration of Undaria pinnatifida fucoidan had no significant effect on the steady-state trough concentrations of letrozole or tamoxifen and was well tolerated. These results suggest that fucoidan in the studied form and dosage could be taken concomitantly with letrozole and tamoxifen without the risk of clinically significant interactions.

Determination of Cotinine, 3′-Hydroxycotinine, and Their Glucuronides in Urine by Ultra-high Performance Liquid Chromatography

The measurement of the primary nicotine metabolites, cotinine and trans-3′-hydroxycotinine, is a useful biomarker of nicotine exposure and metabolism genetics for smoking cessation research. Herein is described an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of these primary nicotine metabolites in urine. Urine samples were diluted one hundred-fold with water and introduced into an ultra-high performance liquid chromatography triple quadrupole mass spectrometer using positive ion electrospray ionization with multiple reaction monitoring. Levels of urinary nicotine metabolites: cotinine, trans-3′-hydroxycotinine, and their respective glucuronides were determined directly using deuterated internal standards and compared with indirect determination by enzymatic hydrolysis. The assay was applied to a community sample of smokers’ urine (n = 280). The assay demonstrated satisfactory performance (relative standard deviation of 1.6–6.5 percent at the 1000 nanograms per milliliter level and >98 percent recovery) suitable for application to smoking studies with a run time less than five minutes. The mean (min-max) levels of cotinine and cotinine-glucuronide were 968 (31-3831) and 976 (9-5607) nanograms per milliliter. The mean (min-max) levels of trans-3′-hydroxycotinine and trans-3′-hydroxycotinine-glucuronide were 3529 (13-21337) and 722 (0-4633) nanograms per milliliter. Direct determination of glucuronide metabolites was superior to indirect measurement using enzymatic hydrolysis, where there was evidence of loss of metabolites during sample preparation. A sensitive and selective ultra-high performance liquid chromatography–tandem mass spectrometry assay was successfully developed for the determination of cotinine, trans-3′-hydroxycotinine, and their glucuronides in urine. The rapid and simple sample preparation makes this assay suitable for high throughput studies involving nicotine metabolism phenotype for both cytochrome P450 2A6 and uridine 5′-diphospho-glucuronosyltransferase, smoking prevalence, and cessation studies.

Gut Microbial Changes, Interactions, and Their Implications on Human Lifecycle: An Ageing Perspective

Gut microbiota is established during birth and evolves with age, mostly maintaining the commensal relationship with the host. A growing body of clinical evidence suggests an intricate relationship between the gut microbiota and the immune system. With ageing, the gut microbiota develops significant imbalances in the major phyla such as the anaerobic Firmicutes and Bacteroidetes as well as a diverse range of facultative organisms, resulting in impaired immune responses. Antimicrobial therapy is commonly used for the treatment of infections; however, this may also result in the loss of normal gut flora. Advanced age, antibiotic use, underlying diseases, infections, hormonal differences, circadian rhythm, and malnutrition, either alone or in combination, contribute to the problem. This nonbeneficial gastrointestinal modulation may be reversed by judicious and controlled use of antibiotics and the appropriate use of prebiotics and probiotics. In certain persistent, recurrent settings, the option of faecal microbiota transplantation can be explored. The aim of the current review is to focus on the establishment and alteration of gut microbiota, with ageing. The review also discusses the potential role of gut microbiota in regulating the immune system, together with its function in healthy and diseased state.

STABILITY OF TIGECYCLINE IN DIFFERENT TYPES OF PERITONEAL DIALYSIS SOLUTIONS

♦ Introduction: Intraperitoneal tigecycline is a potential option for the treatment of peritoneal dialysis (PD)-associated peritonitis caused by microorganisms resistant to commonly used antibiotics. However, the stability of tigecycline must be assessed in the PD solution before evaluating its safety and therapeutic efficacy in PD-associated peritonitis. The objective of this study was to investigate the stability of tigecycline in 3 types of PD solutions at different temperatures for various time points. ♦ Methods: A total of 27 PD bags (9 PD bags for each type of PD solution; 1.5% glucose, 7.5% icodextrin, and 1.5% glucose pH neutral) containing 2 μg/mL of tigecycline were prepared and stored at either 4, 25, or 37°C. An aliquot was withdrawn immediately before (0 hour) and after pre-defined time points. Each sample was analyzed in duplicate for the concentration of tigecycline using a stability-indicating high-performance liquid chromatography (HPLC) technique. Samples were also assessed for pH, color changes, and evidence of precipitation immediately after preparation and on each day of analysis. ♦ Results: Tigecycline in all 3 types of PD solutions retained more than 90% of its initial concentration for at least 216, 72, and 8 hours at 4, 25, and 37°C, respectively. There was no evidence of precipitation at any time under the tested storage conditions. The pH and color of tigecycline admixed PD solutions stored at 4, 25, and 37°C remained essentially unchanged for 336, 96, and 48 hours respectively. ♦ Conclusion: The results obtained from the study provide a platform for future clinical studies aiming to determine the safety and therapeutic efficacy of intraperitoneally administered tigecycline for the treatment of PD-associated peritonitis.

The Role of Epidermal Growth Factor Receptor in the management of Gastrointestinal Carcinomas: Present Status and Future Perspectives.

BACKGROUND The global burden of gastrointestinal cancers, including colorectal, stomach, and esophageal cancers is rising steadily. Several therapeutic approaches have been considered for the treatment of GI carcinomas. However, none showed to halt or cure the disease. There is a need to develop effective targeted molecular therapies; mainly to overcome the adverse effects of currently used treatment regimens, as well as, to benefit a large proportion of cancer patients who do not respond well to chemotherapeutics. METHODS Epidermal growth factor receptor (EGFR) is one of the promising targets for cancer therapy. Through a cascade of events, activation of EGFR plays an important role in the homeostasis and pathogenesis of various disorders, including carcinomas of the gastrointestinal (GI) tract, ranging from oesophagitis to complex colon carcinoma. RESULTS The GI carcinomas are associated with aberrant EGFR expression. In this review, emphasis was made on various EFGR-associated signalling pathways, their mechanisms and role in the formation of gastrointestinal lesions. CONCLUSION The current EGFR-targeting therapeutics and an outline of various novel drug delivery systems that could potentially be employed for targeting EGFR during cancer treatment were discussed. This would help medical, pharmaceutical and other life science researchers in providing broad understanding of the work previously conducted in this field.