Farmaceutische, Biomedische en Dier­geneeskundige Wetenschappen

Doctoraten 2019

Departement Farmaceutische Wetenschappen

What contribute to human exposure to organophosphate esters flame retardants? An experimental approach - Fuchao Xu (16/12/2019)

​Fuchao Xu

  • 16 December 2019
  • Promotoren: Adrian Covaci en Hugo Neels

Abstract

Organofosfaatesters (PFRs) worden gebruikt als alternatieve vlamvertragers (FRs) in de plaats van de verboden polybroomdifenylethers (PBDEs) die negatieve effecten hebben betoond voor de ecosystemen en meer bepaald voor de humane gezondheid. PFRs en gerelateerde FRs worden massaal toegepast in commerciële producten zoals elektronische en elektrische producten, textiel, verven en kunststoffen om brandrisico te beperken. Zij kunnen evenwel gedurende de levensduur van het product in de omgeving worden vrijgegeven. Mensen worden via verschillende routes blootgesteld aan PFRs waaronder inhalatie, huidabsorptie, voeding en stofinname. De hoofddoelstellingen van deze thesis werden enerzijds de verschillende humane opnameroutes van PFRs en andere FRs te onderzoeken en anderzijds de geschikte bemonsteringsstrategieën voor de blootstellingsanalyse te bestuderen. De scriptie onderzoekt ook het mogelijke verband tussen externe (potentiële hoeveelheden, extern te meten)  blootstelling en de interne (reële, gemonitorde hoeveelheden in biologische stalen) van PFRs met behulp van de gegevens van de humane biomonitoring (urine, serum en haar) en de blootstellingsmatrices (lucht, stof, doordrenkte doekjes en voedsel). Op die manier geeft dit werk een diepgaand beeld van de menselijke blootstelling aan PFRs.

In eerste instantie werden analysetechnieken voor externe blootstellingsmatrices en menselijke biologische monsters ontwikkeld en gevalideerd. Naast het optimaliseren van beschikbare analysetechnieken voor lucht, stof en doordrenkte doekjes werd er een nieuwe methode ontwikkeld voor de detectie van FRs in verschillende soorten voedsel op basis van een vloeistofchromatografie gekoppeld aan massaspectrometrie (LC-QqQ-MS). Deze methode laat kostenefficiënte en gelijktijdige analyse toe van PFRs, PBDEs en gehalogeneerde FRs (EHFRs). De snelle en gemakkelijke procedure verbetert ook aanzienlijk de analytische sensitiviteit in vergelijking met bestaande methoden. Om PFRs en hun di-alkyl/aryl- en hydroxylmetabolieten in urine en serum te detecteren werd een biomonitoringsmethode ontwikkeld (LC-QqQ-MS). Deze methoden zijn toegepast in de beoordeling van humane blootstelling aan PFRs en gerelateerde verbindingen in laatstgenoemde studies.

Omdat de meeste studies uitgaan van de stofinname als de belangrijkste humane blootstellingsroute voor FRs werden in deze thesis grondig en vooral nauwkeurig de belangrijkste factoren bij het verzamelen van stofstalen bestudeerd. De resultaten toonden aan dat het FR-profiel in stof aanzienlijk beïnvloed kan worden door lokale/nationale regelgeving, geografische locatie, micro-omgeving, bemonsteringsseizoen en zelfs deeltjesgrootte. Deze elementen demonstreren de noodzaak aan een geschikte stofbemonsteringsstrategie voor de beoordeling van humane blootstelling.

Humane blootstelling aan PFRs in Chinese regio’s met e-waste recycling werd onderzocht als het worst-case blootstellingsscenario. Het verzamelde huisstof had hogere niveaus aan PFR dan stof uit andere landen. Hoewel PBDE-concentraties van stof afkomstig van e-waste recycling gebieden lager noteerden dan PFR-waarden, waren ze nog steeds aanzienlijk hoger dan in normaal huisstof. Lage gehaltes en detectiefrequenties van PFRs werden aangetroffen in lokale scharreleieren wat verwaarloosbaar was in vergelijking met PBDE-waarden in deze eieren. Deze resultaten suggereren dat PFRs zich anders gedragen dan PBDEs en niet bioaccumuleren in eieren, zelfs in een sterk gecontamineerde omgeving. Gezien het belang van eieren als voedingsbron vergeleken met de blootstelling aan stof was de inname van PFRs via  eieren voor lokaal verblijvende mensen echter nog steeds aanzienlijk. Gezondheids- en milieueffecten met betrekking tot e-waste recycling zijn zorgwekkende kwesties.

Deze thesis analyseert ook het uitgebreid onderzoek naar humane blootstelling aan PFRs uitgevoerd in Oslo, Noorwegen. Eenenzestig deelnemers werden gerekruteerd in deze campagne om vloerstof, oppervlaktestof, persoonlijke lucht, stilstaande lucht, doordrenkte doekjes, duplicaatmaaltijden (1 voor de vrijwilliger en 1 voor analyse) te evalueren. Ook urine, serum en haar werden geanalyseerd en gedetailleerde vragenlijsten voor blootstellingsbeoordeling werden beantwoord. PFR-profielen in oppervlaktestof waren vergelijkbaar met vloerstof maar hadden significant hogere gehaltes. PFR-profielen in persoonlijke lucht waren echter volledig verschillend van die in het stilstaande lucht. PFR-waarden in doordrenkte doekjes konden niet worden geassocieerd met gehaltes in lucht en stof. Uit de resultaten bleek dat de selectie van de bemonsteringsstrategie de nauwkeurigheid van de blootstellingsbeoordeling aanzienlijk kan beïnvloeden. Ethylhexyl difenylfosfaat (EHDPHP) was de belangrijkste PFR in het diëet van de Norse deelnemers. Deze blootstellingsroute was goed voor bijna 75% van de totale PFR-blootstelling. Inhalatie en stofinname waren de tweede en de deerde blootstellingsroutes voor de deelnemers. De resultaten toonden aan dat elke PFR zijn individuele belangrijke blootstellingsroute heeft en dat stofopname niet de belangrijkste route is voor alle PFRs. PFR-metabolieten werden gemeten in urine, serum en haar van de deelnemers. Het hoogste aantal metabolieten werd gedetecteerd in de urine. Sommige metabolieten werden geassocieerd met externe blootstellingsmatrices waarvan vloerstof de beste matrix blijkt te zijn om de interne blootstelling van PFRs te voorspellen. Kort samengevat laat deze blootstellingsenquête zien hoe bemonsteringsstrategieën de beoordeling van de humane blootstelling beïnvloeden. De invloed van de verschillende blootstellingsroutes op de PFR concentraties aangetroffen in het cohort uit Noorwegen werd geschetst.

In vitro biological investigation of novel anti-tubercular compound classes and the development of improved research tools - Eveline Torfs (12/12/2019)

Eveline Torfs

  • 12 december 2019
  • Promotoren: Paul Cos en Louis Maes

Abstract

Despite advances in modern medicine and healthcare, tuberculosis (TB) still poses a major threat worldwide. With approximately 10.0 million new cases and the attributable cause of death of an estimated 1.3 million people, TB is part of the top ten leading causes of death worldwide and has been declared the world’s deadliest infectious disease for several years. In addition, the ever-increasing emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains places an enormous strain on current anti-TB chemotherapy. Accordingly, the World Health Organization (WHO) has set up several strategies to improve TB management, in which the discovery and development of novel anti-TB drugs is key. Although the pipeline for novel anti-TB drugs is steadily filling up with novel candidates, the current arsenal is still limted and likely not sufficient due to high attrition rates. Therefore, there is still an urgent need for new anti-TB compounds.

To support and broaden the early discovery phase with knowledge on novel compound classes, the present thesis primarily reports on the in vitro biological evaluation of a series of triazenes, quinones and thiazolidinedione-hydroxamates. The studied compounds were synthesized by chemist of the University of Antwerp (Belgium), University of Ghent (Belgium), University of Ljubljana (Slovenia) and Palacký University (Czech Republik). To evaluate the small compound libraries for antimycobacterial hits and provide a primary evaluation of their in vitro anti-TB properties at the Laboratory for Microbiology, Parasitology and Hygiene (LMPH), an anti-TB drug screening platform was set up. Using this cascade of state-of-the-art assays, several triazene and quinone hits were described in terms of extra- and intracellular antimycobacterial activity, acute cytotoxicity, early genotoxicity and metabolic stability. In addition to the phenotypically identified hits, a small library of target-based synthesized thiazolidinedione-hydroxamates was evaluated. In vitro anti-TB properties of the discovered hits were described and compared to their activity results in an enzymatic assay, which was performed by the Palacký University (Czech Republik).

Secondly, the present thesis reports on the improvement of compound assessment methodologies and development of new research tools as these are equally important as the discovery and development of novel anti-TB drugs. In an effort to improve the standard biosafety level two (BSL2) TB infection model, calcium-alginate encapsulation of Mtb H37Ra was envisaged. Although micro-bead encapsulation was successful, the in vivo course of infection was not enhanced during both the acute and chronic phase of infection. Moreover, the generation of inflammation during infection was not influenced as well. Though, experiments confirmed the disputed statement that Mtb H37Ra is attenuated in mice, rather than avirulent. Further, a next-generation bioluminescent Mtb H37Ra reporter model was developed to facilitate future in vitro and in vivo anti-TB drug screening at the LMPH. Upon optimization of the bioluminescent reaction, the selected reporter was determined to be a sensitive and robust alternative for the prokaryotic luciferase reporter strain currently used in the LMPH’s in vitro anti-TB screening platform. Finally, proof-of-concept studies indicated the presence of a bioluminescent signal in living BALB/c mice, illustrating the novel bioluminescent reporter strain might be used to develop a bioluminescent imaging model (BLI). This would in turn facilitate in vivo anti-TB drug screening at the LMPH.

Phytochemical and analytical research on some medicinal plants from Panama using a metabolomics approach: Cecropia spp. and Crescentia cujete - Andrés Rivera Mondragón (21/11/2019)

​Andrés Rivera Mondragón

  • 21 november 2019
  • Promotoren: Kenn Foubert en Luc Pieters

Abstract

Herbal medicines are described as any form of plant or plant product used in the maintenance of health as well as in the prevention, improvement, diagnosis or treatment of diseases. Due to the growing relevance of these products, we aimed to contribute to the development of scientifically based and quality controlled herbal substances. Some species of the genus Cecropia (Urticaceae) and Crescentia cujete (Bignonaceae) collected in Panama were selected as case study. Although food supplement-derived products from these plant species are commercially available, a comprehensive description of their phytochemical composition and appropriate analytical methods for guaranteeing their quality and safety are still lacking.

A detailed phytochemical investigation on 4 Cecropia species led to the full identification or tentative characterization of 47 compounds, including 2 phenolic acids, 33 flavonoids, 3 flavonolignans and 9 triterpenoid saponins. Among these, two new antiplasmodial flavonolignans were isolated from C. obtusifolia and identified as ent-mururin A and ent-vaccinin A.

The conditions for the extraction of chlorogenic acid, total flavonoids and flavonolignans from leaves of Cecropia species were optimized by using a design of experiment (DOE). A HPLC-DAD method for the quantification of these chemical markers was validated according to the ICH guidelines. The multivariate data analysis of the Cecropia species under investigation revealed the implications for phytochemical analysis in further taxonomic studies.

The gastrointestinal and colonic biotransformation of the crude extract of the leaves of C. obtusifolia, was investigated under in vitro conditions, and the processing and interpretation of results were facilitated by using an automated machine-learning model. This investigation revealed that flavone C-glycosides and flavonolignans were stable throughout their passage in the simulated gastrointestinal tract including the colon phase. On the other hand, the colon bacteria extensively metabolized chlorogenic acid, flavonol and triterpenoid O-glycosides.

An untargeted metabolomics approach combining UPLC-MS/MS-based molecular networking with conventional isolation and NMR methods was carried out for the phytochemical profiling of the fruit pulp of Crescentia cujete. Sixty-six products, including 9 n-alkyl glycosides, 23 phenolic acid derivatives (such as cinnamoyl and benzoyl derivatives), 15 flavonoids, 4 phenylethanoid derivatives and 15 iridoid glycosides were fully or tentatively identified. Among these, 8-epi-eranthemoside, crescentiol A and crescentiol B were reported as three new iridoid glucosides.

In view of future work, the implementation of the validated analytical methods for the quantification of markers in Cecropia spp. and Crescentia cujete will be a great help for chemical standardization and authentication of their commercial derived products.

Formulation development of poorly water-soluble compounds using solubility enhancement methods - Adrienn Baán (19/11/2019)

​Adrienn Baán

  • 19 november 2019
  • Promotoren: Koen Augustyns en Filip Kiekens

Abstract

Formulation development of two poorly water-soluble compounds, UAMC 00523 (developed by the Medicinal Chemistry Laboratory of the University of Antwerp) and mangiferin, has been performed using the following techniques: cyclodextrin complexation, PLGA nanoparticle formulations, amorphous solid dispersions and dry amorphization with mesoporous silica. UAMC 00523 is a non-nucleoside transcriptase derivate with potential activity against Human African Trypanosomiasis. Mangiferin is a natural compound with a broad range of potential health-improving activities including anti-inflammatory, anticancer and antidiabetic effects.

UAMC 00523 was first formulated with different β-cyclodextrin derivatives, with a maximum solubility enhancement factor of 80. This formulation was tested in vivo (performed by the Laboratory of Microbiology, Hygiene and Parasitology of the University of Antwerp) with no detectable effect. Subsequently, PLGA nanoparticle formulations were prepared with different PLGA’s and different preparation conditions. Formulations were tested in a 50 mg/kg concentration in vivo after oral administration with no detectable effect. Next, a metabolic stability study was performed, confirming the lack of in vivo activity due to the extensive metabolic clearance of the compound.

Mangiferin was formulated by spray-drying of PLGA nanoparticles with an entrapment efficiency above 70% but with a low yield (less than 40%) due to the adhesion of particles on the drying chamber and the cyclone. Considering the low solubility of mangiferin in a common solvent with PLGA and the technical limitations, no further development was performed with this technique. Amorphous solid dispersions were also prepared using different HPMC grades as carrier. Partial amorphization of the compound was achieved with increased solubility. The poor solubility of mangiferin limited the further formulation development with solvent-based techniques, therefore a solvent-free method, the dry amorphization with mesoporous silica using a planetary mono mill was further applied. Binary and ternary systems have been prepared using HPMC and Soluplus® as third component in the formulations. By adjusting the milling settings, amorphous samples were prepared in a significantly shorter time than already reported with other carriers. The stability of the samples was evaluated at accelerated stability conditions in open and closed containers. Increased solubility and amorphous stability of 6 months were achieved for the high energy milled sample. The addition of polymer enhanced the amorphization rate of the samples and improved the stability in open and closed conditions.

Design, synthesis and characterization of alpha-amino diaryl phosphonates as serine protease inhibitors with applications in antimicrobial drug discovery - Carlos Moreno Cinos (09/10/2019)

Carlos Moreno Cinos

  • 9 oktober 2019
  • Promotoren: Koen Augustyns en Hans De Winter

Abstract

The serine protease family is the most widely studied group of proteins in biology. These enzymes are of special interest due to their notoriously diverse and well-characterized role in physiological and pathological processes. Under normal circumstances, complex and accurate systems regulate the proteolytic activity of serine proteases. Dysregulation of their function results in pathological disorders.

Diaryl esters of a-amino phosphonates are a group of irreversible inhibitors of serine proteases. Their potent inhibition of serine proteases added to their absolute lack of activity against cysteine or threonine proteases confer them advantage over alternative serine protease inhibitors lacking this selectivity such as chloromethyl ketones, ketoesters or ketoamides. These attributes open for them a broad range of applications in the Medicinal Chemistry field, from the design of small molecules for target inhibition, to the synthesis of chemical tools such as ABPs. The focus of this PhD research lies on these two options.

Design, synthesis and characterization of small inhibitors was undertaken for the antimicrobial drug discovery field. Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress-response and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition and a chemical exploration around the hit compounds was conducted. Altogether 14 new potent inhibitors of E. coli ClpP were identified. Compounds 4.85 and 4.92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.

Utilization of a-amino diaryl phosphonates as chemical tools for the target identification was the second goal of this PhD research, focusing on the design and synthesis of activity-based probes for serine proteases involved in the patho-physiological mechanism of visceral hypersensitivity.

Establishment of an efficient synthetic route with the potential of combining a variety of reporter tags in a last step was achieved. A small library of 12 ABPs with an acceptable inhibitory activity for different subclasses of serine proteases was synthesized.

Development of an in vitro model for human hepatotoxicity using mass spectrometry-based metabolomics - Matthias Cuykx (02/07/2019)

​Matthias Cuykx

  • 2 juli 2019
  • Promotoren: Adrian Covaci, Tamara Vanhaecke en Kris Laukens

Abstract

Abstract doctoraatsthesis Matthias Cuykx - De ontwikkeling van een in vitro model voor humane hepatotoxiciteit door middel van metabolomics op basis van massaspectrometrie

De lever is een multifunctioneel orgaan dat een centrale rol inneemt in de humane fysiologie. De hoge metabole activiteit maakt het orgaan vatbaar voor stress en levertoxiciteit is vaak waargenomen tijdens blootstelling aan chemische producten.

Toxicologie ondervindt een verschuiving naar mechanistisch onderzoek en in vitro technieken als alternatief voor beschrijvende in vivo toxicologie. Metabolomics, de holistische studie van kleine endogene molecules, is de meest actuele weerspiegeling van het fenotype en omvat alle downstream effecten van de respons van het organisme aan een externe stimulus.

Deze thesis is het resultaat van de eerste experimenten aan het Toxicologisch Centrum op het vlak van metabolomics. De resultaten zijn tot stand gekomen door een nauwe samenwerking met de onderzoeksgroep In Vitro Toxicologie en Dermato-Cosmetologie aan de Vrije Universiteit Brussel en de Adrem/Biomina onderzoeksgroep aan de Universiteit Antwerpen. Het doel van de thesis is gefocust op de ontwikkeling van detectieplatformen voor metabolomics toepassingen om het aantal gedetecteerde metabolieten te verhogen en om de data-kwaliteit te verbeteren.

Het ontwikkelen van een betrouwbaar detectieplatform gebaseerd op vloeistofchromatografie en accurate massaspectrometrie omvat een initiële selectie van stationaire en mobiele fases aan de hand van een gekende standaardmix om de impact op de retentieprofielen in te schatten. Verdere optimalisatie met cel-extracten werd toegepast om de scheiding van gelijkaardige metabolieten te verbeteren. Een vergelijking tussen een generieke methode en de ontworpen platformen voor een polaire en apolaire fractie toonde aan dat een optimalisatie het aantal metabole features verdubbelde en de variabiliteit significant reduceerde.

Verbeteringen in de staalvoorbereiding omvatte het gebruik van chamber slides tijdens de celcultuur, de toevoeging van buffers, antioxidanten en chelerende producten tijdens de vloeistof/vloeistofextractie en aanpassingen in de reconstitutieprocedures.

De geobserveerde metabole veranderingen tijdens de blootstelling aan natrium valproaat komen overeen met het typisch metabool profiel van NAFLD beschreven in de literatuur, inclusief uitputting van vrij carnitine gevolgd door een diacyl- en triacylglycerol accumulatie. Blootstelling aan bosentan veroorzaakte bij lage dosissen een carnitine upregulatie, veranderingen in het polyamine metabolisme en activatie van de cel door fosforylatie. Het metaboloom veranderde bij hoge dosissen en omvatten een relatieve triacylglycerol accumulatie ten gevolge van een carnitine depletie, waarschijnlijk omwille van mitotoxiciteit.

Naast gemeenschappelijke metabole veranderingen tijdens blootstelling aan beide toxicanten konden specifieke verschillen in het metaboloom worden geïdentificeerd. Op basis van dit onderscheid kon specifieke informatie verkregen worden om het mechanisme achter de toxiciteit van xenobiotica te verklaren en aldus bestaande adverse outcome pathways ondersteunen en/of verder uitbreiden.

The role of vascular smooth muscle cell contraction and relaxation as active modulators of arterial stiffness - Arthur Leloup (27/05/2019)

​Arthur Leloup

  • 27 mei 2019
  • Promotoren: Gilles De Keulenaer en Paul Fransen

Abstract

Arteriële stijfheid is een toestand waarbij de grote arteriën een verminderde capaciteit hebben om de pulsatiele stroom – die gegenereerd wordt door het linker ventrikel van het hart – te dempen. Het is een belangrijke indicator en initiator van cardiovasculaire, neurovasculaire en renovasculaire aandoeningen. Een significant deel van de arteriële wand bestaat uit gladde spiercellen waarvan de contractiele tonus onder andere gereguleerd wordt door circulerende en lokale factoren. De endotheelcellen aan de binnenkant van de elastische arteriën produceren grote hoeveelheden van het vasodilaterende stikstof monoxide en een verminderde beschikbaarheid hiervan wordt gelinkt aan verhoogde arteriële stijfheid. Hoewel dit suggereert dat de contractiele tonus van de gladde spiercellen een rol speelt in de fysiologische en pathofysiologische regulatie van arteriële stijfheid is de precieze rol van deze ‘actieve’ vaatwandcomponenten nog onduidelijk.

Het primair doel van dit project was om een beter begrip te krijgen van de rol van gladde spiercel contractie en relaxatie als regulatoren van arteriële stijfheid in gezonde condities. Daarnaast werd ook onderzocht of dysfunctie van deze actieve vaatwandcomponenten een rol speelt in de ontwikkeling van acute of chronische vasculaire pathologieën in diermodellen. Onze resultaten bevestigen dat de contractiele tonus van vasculaire gladde spiercellen inderdaad het biomechanische gedrag van gezonde bloedvaten kunnen moduleren, grotendeels bepaald door de basale productie van stikstof monoxide in de endotheelcellen.

Bovendien toonden we aan dat arteriële verstijving die optreedt na angiotensine-II infusie grotendeels het gevolg is van veranderingen in de contractiele tonus van gladde spiercellen in de aorta. Dit bleek niet het geval in een genetisch muismodel met chronische endotheelfysfunctie, wat suggereert dat de ‘actieve’ componenten van de elastische bloedvatwand vooral een belangrijke rol spelen in de vroege stadia van arteriële dysfunctie. Het verbeterde begrip over de bijdrage van de gladde spiercellen en de endotheelcellen tot het bewaren van  een centraal hemodynamisch evenwicht is een belangrijke stap naar de ontwikkeling van nieuwe therapeutische strategieën die zich specifiek richten op de stijfheid van de centrale arteriën.

Oxidative stress and immunity in attention deficit hyperactivity disorder - Annelies Verlaet (24/05/2019)

​Annelies Verlaet

  • 24 mei 2019
  • Promotoren: Nina Hermans en Huub Savelkoul

Abstract

Studies report increased levels of oxidative damage markers and decreased activity of antioxidant enzymes in attention deficit hyperactivity disorder (ADHD). Moreover, ADHD has a high comorbidity with cell-mediated disorders. Though results are not consistent, both oxidative and immune dysbalance may contribute to ADHD by injuring neuronal cells. Hence, due to their antioxidant and immunoregulatory effects, polyphenols appear appropriate in ADHD therapy. One example is Pycnogenol®, an extract from French maritime pine (Pinus pinaster Ait.) bark, but more research is required.

Oxidative stress and immune biomarkers were compared between 57 untreated paediatric ADHD patients and 69 controls: erythrocyte reduced glutathione and plasma lipid soluble antioxidants as markers of antioxidant status, urinary 8-hydroxy-2’-deoxyguanosine and plasma malondialdehyde as oxidative damage markers and plasma cytokines and antibodies as markers of immune status. Though only slightly elevated, an indication of more oxidative damage was found in ADHD. Antioxidant levels however did not differ or were even higher in patients than in the control group, possibly pointing at a compensation mechanism. Slightly higher IgE levels were supported by a trend for more diagnosed allergies in patients than in controls and might point at an immune dysbalance. However, due to the nature of this study, it is unknown whether redox and immune dysbalance play a causative role in ADHD.

Using the Human Embryonic Kidney (HEK) and human monocytic leukaemia (THP-1) cell lines, Pycnogenol® was shown to act as Toll-like receptor (TLR)1/2 and TLR2/6 agonist and TLR5 partial agonist, stimulating pro-inflammatory cytokine secretion and NF-κB activation. Lipopolysaccharide (LPS)-Pycnogenol® complex formation was required to stimulate TLR4. In vitro metabolism of Pycnogenol® caused immuno-suppressive potential against TLR1/2 and TLR2/6, secretion of anti-inflammatory IL-10 and alteration of gut microbial composition, stressing the effect of metabolism on Pycnogenol®’s biological activity.

Finally, a randomised double-blind placebo and active product controlled clinical trial was set up to investigate the efficacy, mechanism of action and value of Pycnogenol® in paediatric ADHD therapy as compared to methylphenidate treatment and placebo, including effects on immunity, oxidative stress and comorbid complaints. Results of this trial, which is currently ongoing, are not available yet.

This thesis indicates that redox and immune dysbalance play a potential role in ADHD aetiology, but decisive evidence remains to be reported. Polyphenol-rich extracts may have a positive impact on ADHD-related behaviour. The ongoing clinical trial will provide further understanding of the pathogenesis and treatment options of ADHD.

In search of predictive biomarkers in cancer immunotherapy – The importance of analytical validation - Kelly Schats (24/04/2019)

​Kelly Schats

  • 24 april 2019
  • Promotoren: Ingrid De Meester en Marc Kockx

Abstract

During the course of the past years new insights in the importance of the immune system in the fight against cancer led to the new area of immunotherapy in the cancer treatment, with positive results in different tumor indications. Unfortunately, immunotherapy does not show positive effects in all patients/tumor entities. This highlights the need for predictive biomarkers allowing patient selection per treatment, which serves both the patient (no unnecessary exposure to drug toxicity) and society (high costs).

The current doctoral thesis highlights the importance of the analytical validation of biomarker tests in both immunohistochemistry (IHC) and mRNA analysis based assays. The programmed cell death ligand 1 (PD-L1) IHC assays and the PanCancer Immune profiling (PCI) panel from Nanostring are studied as an example. Moreover, the challenges for both approaches (respectively discrepancies in staining patterns and different normalization methods) are investigated in detail.

Robust and reproducible results are obtained for 6 different PD-L1 assays as well as for the validation of the PCI panel (Nanostring).

Furthermore, comparison of the 2 laboratory developed test (LDT) PD-L1 IHC assays as well as comparison of the 4 commercial PD-L1 IHC assays on tonsil and tumor tissue showed discrepancies in the PD-L1 staining pattern, mainly for the SP142 assay. Focusing on the commercial PD-L1 IHC assays, the detection system and the epitope of the primary antibodies were found as potential parameters influencing the staining pattern of the PD-L1 IHC assays. Despite the fact that further research is needed, the current results already show that the diagnostic labs should be aware of the impact of the detection system on the performance of the IHC assay. This is of importance when they develop LDT assay to be used patient selection instead of the recommended PD-L1 assays.

Two different published gene signatures were applied on 2 pilot studies in melanoma patients treated with immunotherapy to illustrate the challenges of interpretation and the influence of the different normalization methods. Further evaluation of the genes as single marker revealed some promising genes, irrespective of the applied normalization method, which should be investigated in more detail.

As second part of the multi-analyte testing, protein expression was evaluated using IHC. The most promising results were observed in the Trimixipi study, where the highest AUC values could be observed for the four immune cell characterization markers (CD8, CD20, CD3/FOXP3) together with PD-L1.

Optimizing the spray drying of Lactobacillus rhamnosus GG as an important step towards the development of successful pharmabiotics - Géraldine Broeckx (21/03/2019)

Géraldine Broeckx

  • 21 maart 2019
  • Promotoren: Filip Kiekens en Sarah Lebeer

Abstract

At present time, every hour 80 people die due to the consequences of antimicrobial resistance. A ‘post-antibiotic’ era threatens the global population, as warned by the World Health Organization (WHO). This has prompted the scientific world to search for alternative strategies to manage and prevent diseases. With the increasing awareness of the role that our human microbiome plays in health and disease, one of the strategies explored is the application of probiotics as a preventive and/or therapeutic agent for infectious diseases. Probiotics are defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit to the host’ (Hill et al., 2014).

The road to develop successful pharmabiotics, i.e. probiotics in pharmaceutical formulations, is very challenging. One of the key hurdles for formulators, is to keep the bacteria alive and active during a longer period of time (preferably up to several years). To enhance shelf-life stability, bacteria are dried, which decreases the available free water content, and this slows down the bacterial metabolism. Freeze drying is the commonly used method in the pharmaceutical industry to dry biopharmaceuticals, such as probiotics. However, in recent years, spray drying has been brought forward as a promising alternative to freeze drying. It is a cheap, fast, and continuous process, where the end products consist of a particulate powder. However, due to the high temperatures used during spray drying and the lack of sufficient long-term storage data (to date), the pharmaceutical industry is skeptical to implement this drying technology to process probiotics.

Therefore, the goal of this research is to investigate if spray drying can be used to produce viable probiotics, which can be stored during a sufficient amount of time. Three protection strategies were investigated, namely, addition of protective agents, controlling the process parameters and pretreating the bacterial cells prior to spray drying. Noteworthy, promising results regarding long-term stability (for up to more than two years) were obtained with the addition of small disaccharides (like lactose and trehalose) and the presence of phosphates in the drying medium, even when storing at room temperature.   
Although, the road to develop successful pharmabiotics is still full of hurdles to overcome and problems to be solved, it can be concluded that an exciting future lies ahead for us and our friendly microbes.