Public defence Silvia Molina Bertran 05/12/2023 - Antibacterial and insecticide activities of Persea americana Mill seeds - Department Pharmaceutical Sciences

Promotor: Prof. Paul Cos

DEFENCE ABROAD

Abstract:

In recent decades, resistance to antimicrobials has increased, mainly associated with to overconsumption and the propagation of vectors that transmit microorganisms. In this sense, plant-derived extracts with novel mechanisms of actions are in the focus of several researches. Persea americana Mill is a tree with several known biomedical applications, but pharmacological potential still need to be fully investigated. The current study explores new approaches in the antibacterial activity of ethanolic extracts of P. americana var. americana seeds obtained by the Soxhlet (SE) and Maceration (MaE) methods; being evaluated from the anti-adhesive effects, antibiofilms and the action against the vectors that transmit microorganisms.
Soxhlet methodology was statistically more efficient in the extraction of metabolites with a total solid value of 42.13 ± 1.76 mg/mL compared with MaE (20.46 ± 1.66 mg/mL). Additionally, SE showed a higher amount of polyphenols (5.12 ± 0.18 mg/mL). The phytochemical screening and the UHPLC-DAD-QTOF analysis also showed the presence of polyphenols and neolignan as main compounds identified.
The influence on the post- embryonic development of Musca domestica was demonstrated, especially on the viability of larvae stage. The adulticidal effects of the extracts were determined by LC50 of 2.910 mg/100 mL and 3.944 mg/100 mL for MaE and SE extracts, respectively. Additionally, both extracts showed larvicidal and pupicidal effects against Aedes aegypti, but SE were more active at lower concentrations.
Extracts showed antibacterial activity against Pseudomonas aeruginosa (IC50= 87 and 187 g/mL) and Staphylococcus aureus (IC50= 144 and 159 g/mL). SE and MaE extracts were also able to significantly reduce the bacterial adhesion to A549 lung epithelial cells. Additionally, both extracts inhibited the biofilm growth and facilitated the release of internal cell components in P. aeruginosa, which might be associated with cell membrane destabilization. Real-time PCR and agarose electrophoresis analysis indicated that avocado seed ethanol extracts (64 g/mL) downregulated virulence-related factors such as mexT and lasA genes. Moreover, no significant toxic effects on monocytes, microglia cells and fibroblast human cells were found after treatment (CC50> 500 μg/mL).
The results of this research provide potential value to the reuse of a P. americana Mill var. americana seeds as alternatives to the vector control, thus avoiding microorganisms spreading, and also novelty treatment of infectious diseases. In this sense, bioproducts from avocado seeds can be considered promising anti- adhesive and anti-biofilm agents.

Public defence Ngoc Thao Hien Le 08/11/2023 - Analytical, Antiviral and Chemical Investigations of Selected Classes of Alkaloids - Department Pharmaceutical Sciences

Promotors: Prof. Luc Pieters - Prof. Emmy Tuenter

Location: Aula Q.003, building Q, Campus Drie Eiken

Abstract:

This PhD project focuses on the phytochemical, antiviral and analytical investigation of selected classes of alkaloids. The thesis contains two distinct parts; the first one dealing with Amaryllidaceae alkaloids and potential antiviral agents, more in particular against SARS-CoV-2; and the second one dealing with quality control of alkaloid-containing food supplements based on Maca (Lepidium meyenii).
A general structure elucidation workflow was established, combining the power of computer-assisted structure elucidation (CASE) by DFT (density functional theory) calculations with various chromatographic and spectroscopic techniques. The workflow was successfully applied to determine 2D and 3D structures of all compounds in this study. In addition, the structure of a mistakenly assigned compound named “macaridine” was revised to macapyrrolin C by DFT calculation of chemical shifts.
The phytochemical investigations of the four plant species (Hymenocallis littoralis, Pancratium maritimum, Scadoxus multiflorus, Lepidium meyenii) led to the discovery of nine novel alkaloids as well as extended the phytochemical profiles of the plant species. A library of Amaryllidaceae alkaloids was built and screened in vitro against the emerging SARS-CoV-2 and cytotoxicity on Vero-E6 cell line. As a result, thirteen compounds displayed moderate inhibition of SARS-CoV-2 replication (EC50 = 39 – 100 μM); ten compounds were devoid of activity and cytotoxicity, and twelve compounds lacked selective activity, since they were cytotoxic. Preliminary evaluation of structure-activity relationships revealed four skeleton-types (lycorine, homolycorine, crinine, and galanthamine) as promising scaffolds for further research.
A new UPLC-TQD-MS/MS method was developed and validated for quality control of alkaloids in Maca-containing food supplements, because of their suspected toxicity. This represents the first analytical method for this purpose. Results obtained from analyses of commercial products indicate the alkaloid content of 500 – 600 ppm in pure Maca powder. From a regulatory point of view, and based on safety concerns about the alkaloidal constituents of L. meyenii, it may be advisable to establish a maximum level of particular alkaloids or alkaloid classes in food supplements containing Maca, rather than to request their total absence. Also, we recommend the use of lepidilines as chemical markers for characterization of Maca in all types of commercial preparation due to their high content, exclusive existence in Maca and ultra-sensitivity to MS detection.

Public defence Anastasia Van der Auwera 13/10/2023 - An integrated strategy to characterize anti-inflammatory lead compounds derived from Filipendula ulmaria (meadowsweet) - Department Pharmaceutical Sciences

Promotors: prof. dr. Luc Pieters - prof. dr. Nina Hermans

Location: Aula O.07 building O, Campus Drie Eiken

Abstract:

The scope of this PhD thesis was to develop an integrated strategy, based on natural pro-drugs and their metabolites, to characterize new anti-inflammatory lead compounds derived from Filipendula ulmaria . The plant is widely used in phytotherapy against inflammatory diseases. However, its active constituents are not exactly known. It contains many constituents, such as flavonoid glycosides, which are not absorbed. However, these compounds are metabolized in the colon by gut microbiota, producing potentially active metabolites that can be absorbed. The aim of this study was to characterize the active constituents or metabolites.
As a first step, the phytochemical composition of F. ulmaria was explored in a comprehensive manner. A rich diversity of phenolic constituents was (tentatively) identified via UHPLC-PDA-amMS. In total, 119 compounds were tentatively identified, of which 69 compounds were not reported before in F. ulmaria. In view of the phenolic nature of the main constituents, extensive biotransformation after oral intake before absorption can be expected. This urges the need for identification and activity profiling of the intestinal metabolites. Therefore, as a second step, the comprehensive extract was subjected to in vitro gastrointestinal biotransformation, which mimics the gastric, intestinal and colonic phase, including fecal fermentation in an anaerobic environment. Samples before, during and after biotransformation were analyzed with UHPLC-ESI-QTOF-MS. To tackle the dynamic and complex nature of this data, an in-house automated data analysis workflow for multiclass longitudinal data was used to screen interesting biotransformation profiles in an unbiased manner. Lastly, in vitro anti-inflammatory activity was evaluated by testing the inhibition of NF-κB activation, COX-1 and COX-2 enzyme inhibition. The simulation of gastrointestinal biotransformation showed a decrease in the relative abundance of glycosylated flavonoids such as rutin, spiraeoside and isoquercitrin in the colon compartment, and an increase in aglycons such as quercetin, apigenin, naringenin and kaempferol. The non-biotransformed extract showed significant inhibitory activity in NF-κB. The genuine as well as the metabolized extract showed a better inhibition of the COX-1 enzyme as compared to COX-2. A mix of aglycons present after biotransformation showed a significant inhibition of COX-1. The activity may be explained by an additive or synergistic effect of genuine constituents and metabolites.
To conclude, this new integrated approach offers added value to medicinal plant research, allowing phytochemical identification of compounds and their metabolites after in vitro biotransformation, followed by preliminary in vitro activity testing, omitting time-consuming and expensive in vivo studies in early stages of research.

Public defence Elias Iturrospe 9/10/2023 - Untargeted metabolomics to study ethanol-induced hepatotoxicity in HepaRG cells - Department Pharmaceutical Sciences

Promotors:

Prof. dr. Alexander van Nuijs

Prof. dr. Adrian Covaci

Prof. dr. Tamara Vanhaecke (promotor VUB)

Location: Aula R.04, gebouw R - Campus Drie Eiken

Abstract:

Despite the high prevalence of alcoholic liver disease, its identification and characterization remain poor, especially in early stages such as alcoholic fatty liver disease and alcoholic steatohepatitis. Consequently, this implies diagnostic difficulties, limited therapeutic options and unclear mechanisms of action. Untargeted metabolomics concerns the holistic investigation of endogenous metabolites, low molecular weight (< 1500 Da) biomolecules, that provide information on the biochemical status of biological samples. As the metabolome is located at the most downstream level of cellular organization, metabolomics was used in this thesis in an in vitro set-up to identify biochemical biomarkers able to elucidate the mechanism of ethanol-induced hepatotoxicity. To enable untargeted metabolomics research, analytical platforms were developed using liquid chromatography-quadrupole-time-of-flight high-resolution mass spectrometry (LC-QTOF-HRMS) and coupling to drift tube ion mobility spectrometry was explored. A data analysis workflow was generated, and quality control measures were implemented through the metabolomics workflow.
Excessive ethanol consumption disrupts lipid metabolism and initiates progressive intracellular lipid accumulation, resulting in alcoholic fatty liver disease. HepaRG cells (i.e., a human hepatic cell line) were exposed to ethanol at varying concentrations and durations to mimic this latter phenotype. Distinctive patterns between exposed and control cells were consistently observed, with intracellular upregulation of di- and triglycerides, downregulation of phosphatidylcholines and -ethanolamines, sphingomyelins, and S-adenosylmethionine, among others. Several intracellular metabolic patterns could be related to changes in the extracellular environment, such as increased intracellular hydrolysis of sphingomyelins, leading to increased phosphorylcholine secretion. Carnitine alterations highlighted the interplay between β-oxidation in mitochondria and peroxisomes. The combination of the metabolic fingerprints and footprints enabled a comprehensive investigation of the pathophysiology behind ethanol-induced hepatotoxicity.
To improve in vitro simulation of alcoholic steatohepatitis, HepaRG cells were further exposed to a combination of ethanol and tumor necrosis factor alpha (TNF-α), an inflammation inducer. Co-exposure to TNF-α highlighted its importance in the upregulation of hepatic triglycerides and the downregulation of hepatic phosphatidylcholines and -ethanolamines. In addition, fatty acyl esters of hydroxy fatty acid-containing triglycerides were detected for the first time in human hepatocytes and their alterations showed a potentially important role during the progression of alcoholic steatohepatitis. Ethylated phosphorylcholine was observed as a possible new biomarker of ethanol exposure.
Overall, subjecting the HepaRG liver cell line to metabolomics analyses proved to be a valuable tool to obtain mechanistic insights in ethanol-induced hepatotoxicity. Usage of this tool facilitates future in vivo research by pinpointing interesting biomarkers and metabolic pathways.

Public defence Camilla Scarpellini 20/09/2023 - Identification of novel ferroptosis and necroptosis inhibitors - Optimising the therapeutic potential by improving pharmacokinetic properties - Department Pharmaceutical Sciences

Promotors: Prof. Koen Augustyns - Prof. Hans De Winter

Location: Aula O.05, O-building - Campus Drie Eiken

Abstract:

Cell death can be a consequence of different stress conditions that cells undergo, and it is considered an essential process to control the biological homeostasis of tissues and multicellular organisms. In the past 20 years many novel types of regulated cell death (RCD) have been identified. Particularly, ferroptosis and necroptosis emerged as new types of non-apoptotic forms of cell death involved in different human pathologies such as neurodegenerative diseases, ischemia-reperfusion injury, ocular surface dysfunctions and cardiac diseases. Thus, ferroptosis and necroptosis inhibitors may have therapeutic potential.
This project focused on the design, synthesis, and biological evaluation of novel ferroptosis and necroptosis inhibitors. The goal was to address the phospholipid peroxide accumulation with radical trapping antioxidants (RTAs) for ferroptosis, and to target the activity of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) kinase to suppress necroptosis.
We initiated the ferroptosis segment of the project by utilising UAMC-3203, a novel RTA that was developed within our group. This compound exhibited high potency, stability, solubility and safety in different animal models. To further improve its pharmacokinetics properties, the drug-likeness and permeability, we modified UAMC-3203 introducing different moieties leading to different series of analogues. The majority of the compounds showed high potency in the in vitro assay with an IC50 < 100 nM and in the FENIX assay, a spectrophotometric method to determine lipophilic RTAs potency confirming their anti-ferroptotic potential. UAMC-3844 was the best compound of the series with improved potency and microsomal stability.
In addition, the section on necroptosis started from GSK2656157 (GSK’157), a supposedly specific inhibitor of protein kinase R (PKR)-like ER kinase (PERK), which we previously identified as a much more potent RIPK1 inhibitor. We performed further structural optimisation on the GSK’157 scaffold to develop a novel class of more selective RIPK1 inhibitors. Introducing different substituents on the para-position of the pyridinyl ring we synthesised a series of novel GSK’157 analogues with increased selectivity for RIPK1. The most selective compounds were screened by western blot and tested in vitro for their ability to inhibit RIPK1-dependent apoptosis and necroptosis. The optimisation led to UAMC-3861 as the best compound of this series in terms of activity and selectivity for RIPK1 over PERK. The docking study confirmed our hypothesis therefore leading to a novel series of potent and selective type II RIPK1 inhibitors based on the GSK’157 scaffold.
The most promising candidates, UAMC-3844 and UAMC-3861 can be considered to study ferroptosis and necroptosis driven diseases in vivo.

Public defence Siebe Lievens 05/09/2023 - Exploring the interactions between polyvinyl chloride (micro)plastics and black soldier fly larvae: a biological and a chemical approach - Department Pharmaceutical Sciences

Joint PhD KULeuven - UAntwerpen

Promotors:     prof. dr. Adrian Covaci - dr. Giulia Poma  (UAntwerpen)
                       prof. dr. Mik Van Der Borght - prof. dr. Jeroen De Smet (KULeuven)

Public defence: KU Leuven Campus Geel (Kleinhoefstraat 4, 244 Geel) - Auditorium 2

Abstract:

The increasing global population and demand for animal proteins result in an increased production of feed sources like fish- and soybean meal. This exerts considerable pressure on our planet, due to more deforestation, land and water overuse, and overfishing. Additionally, global municipal waste is also rapidly increasing, consisting for 32 to 50 % of food and greens, which is largely anaerobically digested to produce biogas at present. However, prioritising waste re-utilisation over energy recovery aligns better with the waste management hierarchy and the principles of the circular economy.
One promising solution is rearing insects like black soldier fly (BSF) larvae (Hermetia illucens) on organic waste streams, as they efficiently convert such waste into biomass suitable for the feed industry. However, there are currently knowledge gaps on the safety of BSF larvae as a feed ingredient when reared on organic waste streams containing plastic particles.
Therefore, this PhD research focused amongst others on investigating the interactions between polyvinyl chloride (PVC) (micro)plastics and BSF larvae. The larvae were exposed to substrates containing PVC particles of different sizes to assess the effect on their growth, survival and bioconversion. Results proved that BSF larvae were not significantly affected by the presence of PVC (micro)plastics, while they were not able to degrade the (micro)plastics present in their substrate. Additional tests showed that BSF larvae ingested spherical polyethylene microplastics when the particle size was smaller than their mouth opening (< 110 μm).
Additionally, a chemical approach was used to investigate the bioaccumulation and biotransformation of plastic additives (DINP and DEHT) by BSF larvae. However, this was found to be low, due to the fact that the plastics were too large to be ingested and the migration of the additives to the substrate was limited.
To conclude, it can cautiously be assumed that, from the plastic contamination perspective, BSF larvae can be safely used as feed ingredient when reared on substrates containing PVC (micro)plastics larger than 110 μm

Public defence Katyeny Manuela da Silva 29/08/2023 - Development of mass spectrometry-based bioanalytical platforms for metabolomics

Promotors:

Prof. dr. Alexander van Nuijs

Prof. dr. Adrian Covaci

dr. Matthias Cuykx

Public defence: Tuesday the 29th of August 2023 at 10am in Aula Q002 (Promotiezaal) , building Q, Campus Drie Eiken, Universiteitsplein 1, 2610 Antwerpen (Wilrijk)

Abstract:

Metabolomics is the study of small endogenous molecules (<1500 Da) and their interactions in biological systems (cell culture extracts, plasma, urine, or entire organisms). Alterations in genes, RNA transcripts, and proteins, together with environmental changes, are amplified in the metabolome, reflecting the underlying biochemical activity and state of a system. Untargeted metabolomics, the comprehensive analysis of hundreds of metabolites, has proven to be a powerful hypothesis-generating approach in a wide range of applications such as pharmacology, toxicology, food science, and microbial and plant biotechnology. The unbiased nature of untargeted analysis imposes significant challenges for analytical platform development. The goal of this research was 1) to develop an untargeted metabolomics platform for a broad range of polar metabolites and lipids using state-of-the-art liquid chromatography-high resolution mass spectrometry (LC-HRMS), 2) to explore the potential of ion mobility spectrometry (IMS) as an additional dimension of separation and 3) to improve the confidence of compound annotation by implementing in-house multidimensional libraries (e.g., retention time, MS/MS) using open source tools.
Since one method cannot simultaneously cover the metabolome and lipidome, two complementary workflows were optimized to increase this coverage for different biological matrices. A platform for polar metabolites was developed, comparing different mixed-mode hydrophilic interaction-based stationary phases, mobile phase composition, and instrumental parameters. Lipids are highly diverse compounds, yet their chemical similarities form an analytical challenge for standard LC-MS platforms. Therefore, an additional technique, IMS, was used in addition to LC-MS analysis. The optimized LC-IM-MS method included the use of IM-derived collision cross section (CCS) values; the platform was successfully applied for high-confidence lipid annotation in biological samples.
Further, an easy-to-use workflow for the creation of an in-house metabolite library with curated multidimensional data for 100 metabolite standards was developed using R to generate publicly available data in “.msp” format. Finally, the advantages and current limitations of implementing IMS in LC-HRMS workflows for the analysis of highly isomeric mixtures of oxidized lipids were investigated. The results showed that adding the IM dimension to LC has enormous potential to improve peak capacity. However, a broad dynamic range and fast MS acquisition rates are crucial to separate and annotating isomeric species in biological matrices.

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Public defence Alba Ramos Llorca -27/06/2023 - Design, synthesis and evaluation of novel activity-based probes and inhibitors for trypsin-like serine proteases - Focus on Dry eye disease (DED) and Irritable bowel syndrome (IBS) - Department Pharmaceutical Sciences

Promotors:

Prof. dr. Koen Augustyns

Prof. dr. Pieter Van der Veken

​Tuesday the 27th of June 2023 at 4pm in Aula O.05, building O, Campus Drie Eiken

Abstract:

Proteases catalyze the hydrolysis of peptides or proteins by cleaving peptide bonds. These enzymes are of great interest in biomedical research due to their involvement in many physiological processes. In this thesis, we focus on serine proteases. Serine proteases are involved in several physiological processes, including immune response, cell death, and tissue healing. The upregulation of these proteases can increase inflammatory cytokines, degradation of extracellular matrix components, among others.
We previously obtained an in vivo proof of concept with a multi-target serine protease inhibitor (UAMC-00050) in Dry Eye Disease (DED) and Irritable Bowel Syndrome (IBS). Topical application of this compound in the eye of a tear-deficient dry eye rat animal model reduced both tissue damage and inflammatory parameters. Moreover, UAMC-00050 also causes a decrease in visceral hypersensitivity in a rat model of post-inflammatory visceral hypersensitivity.
The focus of this project was to characterize the proteases involved in DED and IBS. We wanted to use a proteomic technique, activity-based protein profiling (ABPP). ABPP uses chemical probes, known as activity-based probes (ABPs), which react covalently with the active form of the target enzyme. These allow detection, visualization, or affinity purification of the labeled enzymes.
During this project, we developed an extensive library of ABPs with “clickable” affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. For the first time, we demonstrated that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. In further studies, we aim to identify upregulated proteases in DED and IBS biological samples.
Finally, given the promising in vitro results of the ABPs with recombinant enzymes, we developed two new inhibitors targeting trypsin-like serine proteases. The results of their inhibitory potencies for a panel of trypsin-like serine proteases and the first study of their physicochemical properties showed a promising backup compound with the potential to be a therapeutic agent for DED and IBS.

Public defence Tim Boogaerts 22/06/2023 - The role of wastewater-based epidemiology as a complementary information source on lifestyle and health aspects - Department Pharmaceutical Sciences

Promotors:

prof. dr. Adrian Covaci

prof. dr. Alexander van Nuijs

prof. dr. Hans De Loof

Public defence: 22th of June 2023 at 4pm in Aula Q002 (Promotiezaal), building Q, Campus Drie Eiken, Universiteitsplein 1, 2610 Antwerpen (Wilrijk).

Abstract:

Wastewater-based epidemiology (WBE) involves measuring the concentrations of human excretion products (=biomarkers) in influent wastewater (IWW) at different time points and locations in order to evaluate consumption and/or exposure patterns in the general population.
The WBE approach was applied to 24-h composite IWW samples collected from 2019 through 2022 from different Belgian cities, including Antwerp, Boom, Brussels, and Leuven. Different (bio)analytical methods were optimised and validated to detect and quantify concentrations of chemical and biological biomarkers in IWW. Concentrations of biomarkers for stimulant, alcohol, pharmaceutical use and SARS-CoV-2 infections were converted to population-normalised mass loads (PNML) by considering the flow rate and catchment population size. Mobile phone data was used to correct for changing population dynamics. Data was evaluated using a time series based statistical framework to assess temporal changes graphically and quantitatively in the measured PNML.
The developed and validated (bio)analytical methods successfully detected and quantified low concentrations of the biomarkers in IWW from different locations. These methods were also capable of estimating spatio-temporal changes in the PNML of the biomarkers. Therefore, WBE can be useful as a complementary data source to evaluate the impact of governmental interventions on the consumption of different compounds (e.g. drugs, alcohol and pharmaceuticals) and/or exposure to pathogens (e.g. coronavirus). Our findings suggest that stimulant use (i.e. cocaine, amphetamine and ecstasy) was minimally affected by the lockdown measures during the COVID-19 pandemic, while alcohol use decreased in Leuven as a result of the stay-at-home measures. For most psychoactive pharmaceuticals, an increase in the PNML was observed during the lockdown periods in Leuven. The population disruption during the COVID-19 pandemic led to a major change in the socio-demography of the catchment area, resulting in temporal differences in the reported PNML of the biomarkers of opioids and antidepressants. Furthermore, the WBE approach effectively monitored the spread of SARS-CoV-2 infections, mapped possible disease outbreaks and identified the proportion of different known variants of concern within defined population groups.
WBE is able to evaluate the lifestyle and public health of populations with a fast turn-around analysis time and with high spatial and temporal resolution. WBE can also be applied to evaluate the effects of policy changes in different areas and time periods.

Public defence Kimberley Elbrink 14/06/2023 - Application of solid lipid nanoparticles as a drug delivery platform for parenteral and oral administration - Department Pharmaceutical Sciences

Promotor: prof. dr. Filip Kiekens

Location: Wednesday the 14th of June 2023 at 10 am in Aula O.05 building O, Campus Drie Eiken

Abstract:

The development of new drug molecules is an important element of the extended lifespan and improved human health around the globe. Many new drug candidates are highly lipophilic causing solubility and formulation stability issues. This poses a challenge for formulation scientists to develop an optimal drug delivery system for oral and parenteral administration of these drugs. Solid lipid nanoparticles (SLNs) have attracted increasing attention as a promising approach for lipophilic drug delivery. This research focuses on SLNs for parenteral and oral drug delivery to gain more knowledge about the relationship between the formulation and compound and in vitro and in vivo release profiles.
The SLNs were evaluated as a long-acting injectable drug delivery platform for parenteral administration with low and high drug concentrations at equivalent lipid doses. The influence of drug loading and the administration route for the SLNs using two model compounds (bedaquiline and celecoxib) with different physicochemical properties were investigated. Results showed that in vitro drug release was compound-specific, and related to the drug affinity towards the lipid matrix and release medium. The SLNs extended the drug release after administration to rats and the pharmacokinetic parameters were more pronounced for the subcutaneous than the intramuscular administration.
Next, the celecoxib-loaded SLNs were administered orally to rats to determine if the oral bioavailability of the compound dosed in the nanocarriers could function equally when suspended versus solubilized in the lipid matrix. Both formulations offered the potential to improve the compound’s oral bioavailability. This was more pronounced for the solubilized than the suspended compound in the lipid matrix. Moreover, the in vitro release profiles could be used to rank the in vivo bioavailability.
SLNs in aqueous media are subject to physical and chemical instability upon storage. This barrier can be overcome by freeze-drying. The freeze-drying process was optimized through experimental designs and the optimal freeze-dried SLNs were subjected to a 6-month stability study. A successful freeze-drying process was obtained by a rational compromise between a good formulation and optimal conditions in the freezing and drying steps. This yielded an acceptable non-collapsed freeze-dried cake with minimal changes in physicochemical properties, good redispersibility, and 6-month stability at 4 °C for SLNs with trehalose as a cryoprotectant.
In conclusion, this research provided promising results for the parenteral and oral application of SLNs, especially in terms of in vitro and in vivo evaluations, and confirms that SLNs can serve as a versatile platform for poorly water-soluble drugs

Openbare verdediging Christina Christia 14/02/2023 - Karakterisering van voorkomen, metabolisme en bijdrage aan menselijke blootstelling van nieuwe weekmakers aanwezig in het binnenmilieu - Departement Farmaceutische Wetenschappen 

Promotoren: Prof. dr. Adrian Covaci - dr. Giulia Poma

Promotiezaal, gebouw Q - Campus Drie Eiken

Samenvatting:

Recently, several changes have been made to the chemical composition of products used indoors. Legacy chemicals have been replaced by “alternatives” due to evidence for adverse health effects of the former. However, there is still limited information related to the human exposure to these replacement chemicals. Therefore, there is urgent need to investigate their occurrence indoors and the extent of the human exposure to them. In this regard, this study was performed by recruiting twenty-five (n=25) families living in Flanders (Belgium). Two sampling campaigns were organized during winter and summer 2019 in the same households where paired samples of floor dust (n=25), handwipes (n=82) and urine (n=82) from adults and children (aged up to 7 years old) were collected.
Six alternative plasticizers named as, di-propylene glycol dibenzoate (DiPGDB), tri-n-butyl trimellitate (TBTM), bis-3,5,5-trimethyl hexyl phosphate (TMHPh), iso-octyl 2-phenoxy terephthalate (IOPhET), dimethyl azelate (DMA) and dimethyl sebacate (DMS) were detected for the first time in indoor dust after the application of a suspect screening analysis (SSA) workflow. These new plasticizers were then measured in dust using an in-house validated LC-MS/MS analytical method. Based on their levels, the plasticizers DiPGDB, TBTM, IOPhET and TMHPh were further selected to be investigated in handwipes. An innovative new analytical method was applied to the handwipes using a significantly lower amount of sample than reported in literature so far. The external human exposure through inadvertent dust ingestion and dermal absorption were estimated using the concentrations detected in dust and handwipes, respectively.
Finally, to provide an initial assessment of human exposure, the internal exposure to the new plasticizers was assessed for DiPGDB, TBTM and TMHPh, considering the abundancies in dust and handwipes and their theoretical bio-accessibilities. A combined workflow of an in-vitro assay using human liver microsomes (HLMs) and an in-silico prediction using a suitable software was applied and metabolites of Phase I were identified. Together with the predicted ones of Phase II, they were identified in the urine samples. An SSA workflow was employed using LC-QTOF-MS for the identification and semi-quantification.
The plasticizers DiPGDB and TBTM were the dominant plasticizers in dust and handwipes and their metabolites were found in higher levels in human urine. No risk was indicated via the inadvertent ingestion and dermal absorption of these compounds. Dust is a source of these new plasticizers but daily habits (eg. use of personal products) are likely contributing to the overall human exposure.