A combined phase I and II open label study on the effects of a seaweed extract nutrient complex on osteoarthritis

Background: Isolated fucoidans from brown marine algae have been shown to have a range of anti-inflammatory effects. Purpose: This present study tested a Maritech® extract formulation, containing a blend of extracts from three different species of brown algae, plus nutrients in an open label combined phase I and II pilot scale study to determine both acute safety and efficacy in osteoarthritis of the knee. Patients and methods: Participants (n = 12, five females [mean age, 62 ± 11.06 years] and seven males [mean age, 57.14 ± 9.20 years]) with a confirmed diagnosis of osteoarthritis of the knee were randomized to either 100 mg (n = 5) or 1000 mg (n = 7) of a Maritech® extract formulation per day. The formulation contained Maritech® seaweed extract containing Fucus vesiculosis (85% w/w), Macrocystis pyrifera (10% w/w) and Laminaria japonica (5% w/w) plus vitamin B6, zinc and manganese. Primary outcome was the average comprehensive arthritis test (COAT) score which is comprised of four sub-scales: pain, stiffness, difficulty with physical activity and overall symptom severity measured weekly. Safety measures included full blood count, serum lipids, liver function tests, urea, creatinine and electrolytes determined at baseline and week 12. All adverse events were recorded. Results: Eleven participants completed 12 weeks and one completed 10 weeks of the study. Using a multilevel linear model, the average COAT score was reduced by 18% for the 100 mg treatment and 52% for the 1000 mg dose at the end of the study. There was a clear dose response effect seen between the two treatments (P≤0.0005) on the average COAT score and each of the four COAT subscales (pain, stiffness, difficulty with physical activity and overall symptom severity) (P≤0.05). The preparation was well tolerated and the few adverse events were unlikely to be related to the study medication. There were no changes in blood parameters measured over the course of the study with the exception of an increase in serum albumin which was not clinically significant. Conclusion: The seaweed extract nutrient complex when taken orally over twelve weeks decreased the symptoms of osteoarthritis in a dose-dependent manner. It was demonstrated to be safe to use over the study period at the doses tested. The efficacy of the preparation now needs to be demonstrated in a phase III randomized controlled trial (RCT). Australian and New Zealand Clinical Trials Register: ACTRN12607000229471

A combined Phase I and II open-label study on the immunomodulatory effects of seaweed extract nutrient complex

Background: Isolated fucoidans from brown marine algae have been shown to have a range of immune-modulating effects. This exploratory study aimed to determine whether a seaweed nutrient complex containing a blend of extracts from three different species of brown algae plus nutrients is safe to administer and has biological potential as an immune modulator. The study was undertaken as an open-label combined Phase I and II study. Methods: Participants (n = 10) were randomized to receive the study medication at either a 100 mg (n = 5) or 1000 mg (n = 5) dose over 4 weeks. The primary outcome measurement was in vivo changes in lymphocyte subsets. The secondary outcome measures were ex vivo changes in T-lymphocyte (CD4 and CD8) activation, phagocytosis of granulocytes and monocytes, T helper 1/T helper 2 cytokines, and serum oxygen radical absorbance capacity. Results: The preparation was found to be safe over the 4 weeks at both doses tested. There were no clinically relevant changes to blood measurements of hemopoietic, hepatic, or renal function. Immunomodulatory measurements showed no dose response between the two doses. The combined results from the two doses demonstrated a significant increase in cytotoxic T cell numbers and phagocytic capacity in monocytes, and a significant decrease in levels of the inflammatory cytokine interleukin 6. A separate analysis of the 100 mg dose (n = 5) alone showed a significant linear component over time (P \u3c 0.05) for phagocytosis by both granulocytes and monocytes. Conclusion: The seaweed nutrient complex was safe to use when taken orally over 4 weeks. The preparation was demonstrated to have potential as an immune modulator, and this bioactivity deserves further exploration

A forced titration study of the antioxidant and immunomodulatory effects of Ambrotose AO supplement

Background Oxidative stress plays a role in acute and chronic inflammatory disease and antioxidant supplementation has demonstrated beneficial effects in the treatment of these conditions. This study was designed to determine the optimal dose of an antioxidant supplement in healthy volunteers to inform a Phase 3 clinical trial. Methods The study was designed as a combined Phase 1 and 2 open label, forced titration dose response study in healthy volunteers (n = 21) to determine both acute safety and efficacy. Participants received a dietary supplement in a forced titration over five weeks commencing with a no treatment baseline through 1, 2, 4 and 8 capsules. The primary outcome measurement was ex vivo changes in serum oxygen radical absorbance capacity (ORAC). The secondary outcome measures were undertaken as an exploratory investigation of immune function. Results A significant increase in antioxidant activity (serum ORAC) was observed between baseline (no capsules) and the highest dose of 8 capsules per day (p = 0.040) representing a change of 36.6%. A quadratic function for dose levels was fitted in order to estimate a dose response curve for estimating the optimal dose. The quadratic component of the curve was significant (p = 0.047), with predicted serum ORAC scores increasing from the zero dose to a maximum at a predicted dose of 4.7 capsules per day and decreasing for higher doses. Among the secondary outcome measures, a significant dose effect was observed on phagocytosis of granulocytes, and a significant increase was also observed on Cox 2 expression. Conclusion This study suggests that Ambrotose AO® capsules appear to be safe and most effective at a dosage of 4 capsules/day. It is important that this study is not over interpreted; it aimed to find an optimal dose to assess the dietary supplement using a more rigorous clinical trial design. The study achieved this aim and demonstrated that the dietary supplement has the potential to increase antioxidant activity. The most significant limitation of this study was that it was open label Phase 1/Phase 2 trial and is subject to potential bias that is reduced with the use of randomization and blinding. To confirm the benefits of this dietary supplement these effects now need to be demonstrated in a Phase 3 randomised controlled trial (RCT)

Transcutaneous electrical nerve stimulation and chronic intractable angina pectoris

The treatment trial being reported investigated whether the frequency and severity of pain in 11 patients with chronic intractable angina pectoris were decreased by TENS applied adjacent to T and T spinous processes. Pulse and blood pressure were measured before, during and after each treatment. Results showed a significant decrease in frequency and severity of pain. Rate pressure product was calculated to try to identify the mode of action. The mean RPP before TENS application, compared with the mean RPP at 30 minutes and at 90 minutes, was significantly reduced. It was concluded from these results that TENS should be considered as an adjunct to medical management in those patients with angina pectoris not satisfactorily controlled by optimal medication

The other side of the coin: safety of complementary and alternative medicine

Most consumers consider complementary and alternative medicine (CAM) products inherently safe. The growing simultaneous use of CAM products and pharmaceutical drugs by Australian consumers increases the risk of CAM-drug interactions. The Therapeutic Goods Administration (TGA) has a two-tier, risk-based regulatory system for therapeutic goods - CAM products are regulated as low risk products and are assessed for quality and safety; and sponsors of products must hold the evidence for any claim of efficacy made about them. Adverse reactions to CAM products can be classified as intrinsic (innate to the product), or extrinsic (where the risk is not related to the product itself, but results from the failure of good manufacturing practice). Adverse reactions to CAM practices can be classified as risks of commission (which includes removal of medical therapy) and risks of omission (which includes failure to refer when appropriate). While few systematic studies of adverse events with CAM exist, and under-reporting is likely, most CAM products and practices do not appear to present a high risk; their safety needs to be put into the perspective of wider safety issues. A priority for research is to rigorously define the risks associated with both CAM products and practices so that their potential impact on public health can be assessed

Vascular mechanisms in osteoarthritis

Superficial injury and fibrillation of articular cartilage as a consequence of ageing, genetic, hormonal or mechanical factors are not necessarily associated with joint pain. However, failure of joint cartilage accompanied by synovitis and abnormalities in subchondral bone and its vasculature generally is, the syndrome being known as osteoarthritis. We suggest that the progression of early cartilage fibrillation of symptomatic OA arises initially as a consequence of the release into synovial fluid of cartilage-derived antigens that activate joint lining macrophages and circulating leukocytes, thereby establishing a synovitis. Pro-inflammatory mediators and pro-coagulant factors etc. not only perpetuate cartilage destruction but also promote a state of hypercoagulation, hypofibrinolysis, thrombosis and ischaemic bone necrosis at compromised sites such as in the subchondral vasculature. These events are augmented by ageing and associated hormonal changes. On the basis of this hypothesis we suggest that anti-thrombotic/anti-lipidaemic agents that also exhibit anti-inflammatory activity could be effective anti-osteoarthritic drugs. Experimental studies are described which support this proposal

Insights into catalytic mechanism of two novel LPMOs for lignocellulose degradation

160 σ.Η παραγωγή βιοκαυσίμων, και συγκεκριμένα βιοαιθανόλης, από λιγνινοκυτταρινούχο βιομάζα αποτελεί το κέντρο επιστημονικού και βιομηχανικού ενδιαφέροντος τα τελευταία χρόνια. Συγκεκριμένα, για να αποκτήσει βιώσιμο χαρακτήρα η παραγωγή 2ης γενιάς βιοαιθανόλης, η προσπάθεια έχει επικεντρωθεί στη μείωση του ενζυμικού φορτίου και, κατά συνέπεια, του κόστους που προκύπτει κατά τη διαδικασία μετατροπής της βιομάζας σε ζυμώσιμα σάκχαρα. Σκοπός της παρούσας εργασίας ήταν η μελέτη του μηχανισμού κατάλυσης δύο καινοτόμων υδατανθρακικών μονοοξυγενασών ((Lytic) Polysaccharide Monooygenases, (L)PMOs ) από του μύκητες Sporotrichum thermophile και Fusarium oxysporum. Για το λόγο αυτό κλωνοποιήθηκαν και εισήχθησαν με επιτυχία στο γονιδίωμα της μεθυλότροφης ζύμης – ξενιστή Pichia pastoris τα αντίστοιχα γονίδια. Και οι δύο πρωτεΐνες περιείχαν το φυσικό σηματοδοτικό πεπτίδιο, με στόχο κατά την έκφρασή τους να εμφανίζουν τη φυσική αλληλουχία στο αμινοτελικό τους άκρο, η οποία συμμετέχει στο συντονισμό του ιόντος χαλκού, που βρίσκεται στο ενεργό τους κέντρο. Πραγματοποιήθηκε βιοπληροφορική ανάλυση, κατά την οποία επιχειρήθηκε η κατάταξη των δύο PMO ανάλογα με την ομολογία που δείχνουν, με τις κατηγορίες PMO που υπάρχουν στη βιβλιογραφία. Έτσι από τα χαρακτηριστικά της αλληλουχίας τους, κατατάχθηκαν στην κατηγορία PMO 3, οι οποίες φαίνεται να οξειδώνουν το γλυκοζιδικό δεσμό στη θέση 1 ή/και στη θέση 4. Μετά την επιτυχή έκφραση και καθαρισμό των δύο PMO, εξετάστηκε η ικανότητά τους να αποδομούν μια ποικιλία σακχαρούχων υποστρωμάτων. Η FoCel61 Native N – terminal εμφάνισε ενεργότητα σε πολυσακχαρίτες όπως η β – γλουκάνη κριθαριού και λιχενάνη, αλλά και σε κελλο-ολιγοσακχαρίτες, όπως η κελλοτετραόζη και η κελλοπενταόζη. Η StCel61 Native N – Terminal δεν εμφάνισε αντίστοιχη ενεργότητα στα υποστρώματα αυτά. Στα υποστρώματα που ανιχνεύθηκε ενεργότητα της FoCel61 Native N –terminal, μελετήθηκε η επίδραση της παρουσίας αναγωγικών παραγόντων όπως του ασκορβικού οξέος, και της δεϋδρογονάσης της κελλοβιόζης (CDH, Pycnoporus cinnabarinus) στη δράση του ενζύμου. Ωστόσο, κανένας από τους αναγωγικούς παράγοντες δεν ενίσχυσε τη δράση της FoCel61 Native N – terminal. Δείγματα από τα πειράματα ανίχνευσης ενεργότητας αναλύθηκαν με τη βοήθεια υγρής χρωματογραφίας ανιονανταλλαγής (HPAEC) με σκοπό την ανίχνευση και ταυτοποίηση των υδατανθρακικών προϊόντων από την αποδόμηση των υποστρωμάτων. Η αδυναμία ανίχνευσης οξειδωμένων προϊόντων στη θέση 1, όπως γλυκονικού οξέος, σε συνδυασμό με την παραγωγή αναγωγικών ολιγοσακχαριτών υποδεικνύει ότι η FoCel61 Native N – terminal οξειδώνει τον άνθρακα στη θέση 4 κατά την διάσπαση του γλυκοζιδικού δεσμού. Κατόπιν εξετάστηκε η συμβολή των ενζύμων αυτών στην αποικοδόμηση λιγνινοκυτταρινούχου υποστρώματος, όπως έλατο το οποίο έχει υποστεί επεξεργασία με ατμό υπό ήπιες όξινες συνθήκες. Από τα πειράματα, διαπιστώθηκε η συνεργιστική δράση του συστήματος StCel61 Native N – Terminal – κλασσικών κυτταρινασών. Η μελέτη επεκτάθηκε και στην StCel61, η οποία περιέχει κάποια επιπλέον αμινοξέα στο αμινοτελικό της άκρο, και η οποία εξετάστηκε σε παλαιότερη έρευνα (Δημαρόγκωνα, 2012). Τα αποτελέσματα έδειξαν πως τα δυο ένζυμα εμφανίζουν εφάμιλλη συνεργιστική δράση όταν προστεθούν σε μίγμα με κλασσικές κυτταρινάσες, αποδεικνύοντας πως η παρουσία αμινοξέων στο αμινοτελικό άκρο των PMO δεν επηρεάζει την ενεργότητά τους. Η FoCel61 Native N – terminal δεν κατάφερε να ενισχύσει την αποδόμηση του λιγνινοκυτταρινούχου υλικού από τις κυτταρινάσες. Τέλος επιχειρήθηκε η αποδόμηση προκατεργασμένου άχυρου σίτου (PWS), από την StCel61 Native N – terminal σε πιλοτικό αντιδραστήρα ρευστοποίησης, όπως αυτή εκκρίθηκε στο εξωκυτταρικό υγρό της καλλιέργειας (crude). Ωστόσο, δεν μπόρεσε να ανιχνευθεί καμιά επίδραση της PMO στο ιξώδες του PWS, το οποίο χρησιμοποιήθηκε ως μέτρο της αποδόμησης του υποστρώματος.Lately, the production of biofuels, and especially bioethanol, from lignocellulosic biomass has been of high scientific and industrial interest. Specifically, in order for the production of second generation bioethanol to become sustainable, the effort is focused on the reduction of enzymatic load and, and as a result, the cost derived from the conversion of biomass into fermentable sugars. The main purpose of this thesis was to study the catalytic mechanism of two novel (lytic) polysaccharide monooxygenases ((L)PMOs) from Sporotrichum thermophile and Fusarium oxysporum. LPMOs are recently discovered biocatalysts which cleave oxidatively polysaccharide substrates. StCel61 and FoCel61 were cloned and heterologously expressed in the methylotrophic yeast Pichia pastoris. Both enzymes were expressed with their natural signal peptide, in order to be secreted with the naturally occuring N – terminal amino acid, which is involved in active site copper coordination. Bioinformatic analysis was carried out, so as to classify the examined enzymes into one of the three PMO categories described in recent literature (Vu et al). Multiple sequence alignment showed that these proteins share the highest homology with PMO3 enzymes, which hydroxylate position C1 and/or C4 of the glucose ring. After the successful expression and purification of both PMOs, their ability to oxidatively cleave a variety of polysaccharide substrates was examined. FoCel61 Native N – terminal showed activity on polysaccharide substrates, such as barley β – glucan and lichenan, but also on cellooligosaccharides, such as cellotetraose and cellopentaose. No activity on these substrates was detected in the case of StCel61 Native N – terminal. The boosting effect of reducing agents such as ascorbic acid and cellobiose dehydrogenase (CDH from Pycnoporus cinnabarinus) on FoCel61 Native N - terminal was also investigated. However, no significant impact on the activity of the examined enzyme was detected. Samples from enzyme activity experiments were analyzed with anion exchange chromatography (HPAEC), in order to detect and identify the carbohydrate products of substrate degradation. The failure to detect gluconic acid in the hydrolyzed products, in combination with the release of oligosaccharides with free reducing ends, indicates that FoCel61 Native N – terminal is specific for C4 oxidation. Subsequently, the contribution of these enzymes into the degradation of lignocellulosic material, such as steam pretreated under weak acidic conditions spruce (PS), was examined. The experimental results confirmed the synergistic effect of StCel61 native N – terminal on common cellulases. This experiment was also performed for StCel61, the same LPMO expressed in a previous work using a different cloning strategy that resulted in a recombinant enzyme with a small number of additional residues at the N – terminal (Dimarogona, 2012). The detected synergism effect was similar, indicating that the presence of additional amino – acids in the PMO N – terminal does not severely affect enzyme function. As far as FoCel61 Native N – terminal is concerned, it could not boost the degradation of lignocellulosic substrate by common cellulases. Finally, biomass decomposition experiments were carried out on pretreated wheat straw (PWS) in the presence of crude StCel61 Native N – terminal, in a pilot liquefaction reactor. No effect of LPMO activity could be detected on PWS viscosity, which was measured as an indicator of substrate depolymerization.Βασίλειος Π. Χέρα