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Personalized or individualized medicine is considered as an innovative approach to deliver the right dose to the right person at the right time. However, current technologies producing pharmaceutical products do not address the growing need for these new therapies. The aim of this work was the preparation of long-acting implantable dosage forms in which the drug release is regulated by the polymer matrix and the design of the implant. Paliperidone palmitate (PP), was selected as model drug. Indeed, PP is sensitive to thermal degradation above its melting temperature and its amorphous state remains difficult to obtain and stabilize. The first part of this study focused on obtaining and stabilizing the amorphous form of PP. A ternary mixture based on two polymers was therefore selected to be printed as dosage forms. Following an in vitro dissolution test, different release profiles were obtained depending on the selected printing parameters. Then, the stability of the amorphous state of PP was assessed at two different temperatures. During this test, implants were placed at 4°C and 25°C ± 40% residual humidity for 3 months. In the 2nd part, a 3D printing technology reducing the number of steps required to produce a suitable formulation was studied. This technology was based on a pressure extrusion-based printing process. Polycaprolactone was chosen as biocompatible and bioresorbable polymer matrix. Two different designs have been studied, namely a ring and a disk. The goal was to compare the effect of the design on the release of PP. An in vitro dissolution test was performed over a period of 3 to 6 months. In the last part, the development of a proof of concept of a predictive model taking into account the design of the printed form was studied. The impact of different printing parameters on the drug release were studied through a Design of Experiments (DoE). Finally, a second DoE was used to predict the release of PP from complex dosage forms combining two formulati, Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie), info:eu-repo/semantics/nonPublished

Among the different pulmonary fungal infections, aspergillosis, and in particular invasive pulmonary aspergillosis (IPA), are becoming the most worrying diseases in immunocompromised patients. This is due to their high incidence and mortality. Indeed, invasive aspergillosis manifests as invasive pulmonary disease accounting for 50/60% of all cases, with a mortality of 50-90% in severely immunocompromised patients. Triazoles act by inhibiting 14-α demethylase, a fungal cytochrome P450 enzyme implicated in the synthesis of ergosterol, an essential constituent of fungal cell walls. Moreover, they interact with the same cytochrome present in large quantities in the human liver, inducing possible drug-drug interactions in IPA patients. Consequently, interactions resulting from inhibitors, inductors, or substrates of cytochromes can modify the plasmatic concentrations of triazoles or other drugs administered concomitantly. To overcome these important issues, pulmonary delivery of triazoles could be an interesting alternative to conventional routes.The aim of this work was to develop triazole-based dry powders for inhalation able to be deposited adequately in the lungs, with a release of drug and a lung retention that can optimize its pharmacological action. This work focused on two active pharmaceutical ingredients (API): itraconazole (ITZ), for which improved solubility was needed, and voriconazole (VCZ), for which slow release was required.Concerning ITZ, solid dispersions for inhalation (SDIs) comprising ITZ and mannitol were previously developed in our laboratory. The selected SDI showed interesting results in terms of improved dissolution and lung retention in vivo in mice during a pharmacokinetic study. Therefore, this SDI was tested in a murine preclinical model of IPA and showed promising results in terms of prophylaxis efficacy. One aim of this work was to continue the pharmaceutical development of this promising SDI by making a scaling-up study. These methods we, Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie), info:eu-repo/semantics/nonPublished

This PhD thesis aimed at developing synergistic combinations of antibiotics in the form of dry powder for inhalation to be administered to patients with Cystic Fibrosis (CF). The combinations comprise of a macrolide, used in patients with CF for its anti-inflammatory effetcs, and an antibiotic active against Pseudomonas aeruginosa, the main pathogen causing chronic lung infection in patients with CF. The PhD thesis was divided into three different studies. First, microbiological study, examining antimicrobial resistance in CF population to different antipseudomonals and macrolides. Second, pharmacological study, focusing on the development of biofilm infection model relevant to CF pathophysiology and the activity of antibiotics alone or in combination in this model. Third, pharmaceutical study, involving the analytical and formulation development(laboratory and industrial scale) for the Tobramycin-Clarithromycin combination to be used as a dry powder for inhalation. This work highlights the importance of achieving high local concentrations of antibiotics in the lung and provides a candidate formulation for preclinical and clinical development of a combination of antibiotics for the treatment of chronic respiratory infection due to P. aeruginosa in patients with CF., (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2017

Cystic fibrosis (CF) patients receive chronic treatment with macrolides for their antivirulence and anti-inflammatory properties. We, however, previously showed that Pseudomonas aeruginosa, considered as naturally resistant to macrolides, becomes susceptible when tested in a eukaryotic medium rather than a conventional broth. We therefore looked for specific macrolide resistance determinants in 333 CF isolates from four European CF centres in comparison with 48 isolates from patients suffering from hospital-acquired pneumonia (HAP). Minimum inhibitory concentrations (MICs) of macrolides and ketolides measured in eukaryotic medium (RPMI-1640) were higher towards CF than HAP isolates. Gene sequencing revealed mutations at three positions (2045, 2046 and 2598) in domain V of 23S rRNA of 43% of sequenced CF isolates, but none in HAP isolates. Enzymes degrading extracellular polymeric substances also reduced MICs, highlighting a role of the mucoid, biofilm-forming phenotype in resistance. An association between high MICs and chronic azithromycin administration was evidenced, which was statistically significant for patients infected by the Liverpool Epidemic Strain. Thus, ribosomal mutations are highly prevalent in CF isolates and may spread in epidemic clones, arguing for prudent use of oral macrolides in these patients. Measuring MICs in RPMI-1640 could be easily implemented in microbiology laboratories to phenotypically detect resistance.

Lung cancer is the deadliest cancer in the world, with a global 5-year survival rate of about 15%. Despite a notable impact of the latest improvements in prevention, screening, detection and staging, the efficacy of conventional treatments is not sufficient and has reached a therapeutic plateau. These conventional treatments involve a combination of surgery, radiotherapy (RT) and chemotherapy (CT). CT is used in almost all stages: in operable and inoperable stages to limit tumour cell invasion and in latest stages as a palliative treatment. Cisplatin is one of the most frequently used and most potent drugs available. It is administered by parenteral route at doses limited by its high and cumulative nephrotoxicity but also by other systemic toxicities (e.g. ototoxicity). Its administration therefore requires many precautions (long hydration procedure, surveillance of the renal function), which mobilize medical personnel. A major limitation of parental CT is the low concentration of drug that successfully reaches the tumour or the metastases. A potential additional modality could be aerosolized CT to localize lung cancer treatment. It has shown a relative local tolerance for cisplatin through preclinical and clinical studies in humans by means of nebulized solutions or liposomal formulations. As a local treatment, aerosolized CT has a clear pharmacokinetic (PK) advantage, as it can increase local exposure while decreasing systemic exposure. However, because CT drugs, such as cisplatin, are active at rather high doses (in the mg range), the duration of administration from nebulizers is very long as it depends on the drug solubility or on drug encapsulation into liposomes. They also pose a high risk of environmental contamination and require HEPA-filtrated hoods during the nebulization procedure. Of all the inhalation devices available to deliver high drug doses, dry powder inhalers (DPIs) were chosen in this work. These were chosen to circumvent the above issues by pro, Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie), info:eu-repo/semantics/nonPublished

Cystic fibrosis (CF) patients receive chronic treatment with macrolides for their antivirulence and anti-inflammatory properties. We, however, previously showed that Pseudomonas aeruginosa, considered as naturally resistant to macrolides, becomes susceptible when tested in a eukaryotic medium rather than a conventional broth. We therefore looked for specific macrolide resistance determinants in 333 CF isolates from four European CF centres in comparison with 48 isolates from patients suffering from hospital-acquired pneumonia (HAP). Minimum inhibitory concentrations (MICs) of macrolides and ketolides measured in eukaryotic medium (RPMI-1640) were higher towards CF than HAP isolates. Gene sequencing revealed mutations at three positions (2045, 2046 and 2598) in domain V of 23S rRNA of 43% of sequenced CF isolates, but none in HAP isolates. Enzymes degrading extracellular polymeric substances also reduced MICs, highlighting a role of the mucoid, biofilm-forming phenotype in resistance. An association between high MICs and chronic azithromycin administration was evidenced, which was statistically significant for patients infected by the Liverpool Epidemic Strain. Thus, ribosomal mutations are highly prevalent in CF isolates and may spread in epidemic clones, arguing for prudent use of oral macrolides in these patients. Measuring MICs in RPMI-1640 could be easily implemented in microbiology laboratories to phenotypically detect resistance., SCOPUS: ar.j, info:eu-repo/semantics/published

Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. This study compares the antimicrobial susceptibility of 153 P. aeruginosa isolates from the United Kingdom (UK) (n=58), Belgium (n=44), and Germany (n=51) collected from 120 patients during routine visits over the 2006-2012 period. MICs were measured by broth microdilution. Genes encoding extended spectrum β-lactamases (ESBL), metallo-β-lactamases and carbapenemases were detected by PCR. Pulsed Field Gel Electrophoresis and Multi-Locus Sequence Typing were performed on isolates resistant to ≥ 3 antibiotic classes among penicillins/cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, polymyxins. Based on EUCAST/CLSI breakpoints, susceptibility was ≤ 30%/≤ 40% (penicillins, ceftazidime, amikacin, ciprofloxacin), 44-48%/48-63% (carbapenems), 72%/72% (tobramycin), and 92%/78% (colistin) independently of patient's age. Sixty percent of strains were multidrug resistant (MDR; European Centre for Disease prevention and Control criteria). Genes encoding ESBL (most prevalent BEL, PER, GES, VEB, CTX-M, TEM, SHV, and OXA), metallo β-lactamases (VIM, IMP, NDM), or carbapenemases (OXA-48, KPC) were not detected. The Liverpool Epidemic Strain (LES) was prevalent in UK isolates only (75% of MDR isolates). Four MDR ST958 isolates were found spread over the three countries. The other MDR clones were evidenced in ≤ 3 isolates and localized in a single country. A new sequence type (ST2254) was discovered in one MDR isolate in Germany. Clonal and non-clonal isolates with different susceptibility profiles were found in 21 patients. Thus, resistance and MDR are highly prevalent in routine isolates from 3 countries, with carbapenem (meropenem), tobramycin and colistin remaining the most active drugs., SCOPUS: ar.j, info:eu-repo/semantics/published

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Introduction: Efficient Itraconazole (ITZ) oral administration shows numerous limitations because of (i) the poor aqueous ITZ solubility inducing low and unpredictable bioavailability, (ii) the hepatic metabolism to an active metabolite and (iii) the large cytochrome inhibition increasing risks of drug-drug interactions. In case of pulmonary fungal infections, these issues increase the variability of plasmatic concentration in lungs to combat or prevent infection. ITZ pulmonary delivery would be an alternative by increasing ITZ concentration in the lungs while decreasing systemic exposure. Industrial production requires high solids/solutes concentration, using easily evaporable and non-flammable solvents (water, ethanol, isopropanol, ethyl acetate, etc.) while keeping dry powder properties obtained (particles size, residual moisture) at the laboratory scale to reach lungs but at a higher production scale.Materials and method: A screening of ITZ and mannitol solubility is performed on different organic solvent mixtures. Solid dispersions containing ITZ in mannitol matrix were obtained from solutions (So1, So2, and So3) or suspension (Su1) at 3.0% (So1, So3), 4.5% (So2) or 8% (Su1) (w/v) in the preferential organic solvent mixture. In the solutions, mannitol and 35% (So1, So2) or 60% (So3) of ITZ were dissolved. In the suspension, 10% of ITZ were dissolved and some particles of mannitol were suspended. The production method was the spray-drying using a B-290 Mini Spray Dryer (Büchi, Switzerland). The powders were characterized on their physico-chemical and aerodynamic properties (fine particle fraction - FPF), and on their dissolution profiles. Results: All the solid dispersion-based dry powders obtained after spray-drying showed no presence of crystalline ITZ (i.e. complete amorphous state). Because of drying of dissolved particles in the presence of undissolved mannitol particles, solid dispersion from the suspension presented median size (d(0.5)) bigger than solid, info:eu-repo/semantics/nonPublished

Compte tenu de ses aspects multiples, de sa dangerosité potentielle et du taux desurvie considérablement bas qui lui est associé dans ses formes les plus graves, l’aspergillosepulmonaire est encore à l’heure actuelle dévastatrice sur le plan clinique. L’approchemédicamenteuse conventionnelle consiste en l’administration par voie orale ouintraveineuse (IV) d’agents antifongiques. Ces voies classiques requièrent l’administration dedoses très élevées qui sont nécessaires à l’obtention de concentrations systémiquessuffisantes pour obtenir un effet thérapeutique au niveau pulmonaire. Cependant, cesconcentrations systémiques sont également la cause d’effets secondaires indésirables etd’interactions médicamenteuses importantes. Une alternative thérapeutique à ces voiesclassiques serait de localiser ces antifongiques dans le poumon, en utilisant la voie inhalée.Cela permettrait d’augmenter le taux de succès thérapeutique en déposant et enconcentrant directement la dose au niveau du site d’infection tout en minimisant lesconcentrations systémiques.Pour ce faire, nous avons choisi de développer des poudres sèches pour inhalation àbase d’itraconazole (ITZ), un antifongique actif à l’égard des souches d’aspergillus. Celles-cisont administrable via un inhalateur à poudre sèche pour les avantages que présente cemode d’administration comparativement aux nébuliseurs et aux inhalateurs pressurisés. Ledéveloppement des formulations implique entre autres l’obtention de caractéristiquesaérodynamiques appropriées, c’est-à-dire, ayant, après décharge à partir d’un dispositifd’inhalation, un profil de déposition pulmonaire permettant d’atteindre des dosespulmonaires pharmacologiquement efficaces. Toutefois, l’ITZ présente une solubilitéaqueuse extrêmement faible (solubilité aqueuse à pH 7 ~ 4 ng/ml à 25°C). Or, une foisdéposée dans le poumon, la dose inhalée doit se solubiliser pour exercer son actionpharmacologique. Nou, Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/nonPublished

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Christophe Duret,1 Nathalie Wauthoz,1 Thami Sebti,2 Francis Vanderbist,2 Karim Amighi11Laboratoire de Pharmacie Galénique et de Biopharmacie, Université Libre de Bruxelles, Brussels, Belgium; 2SMB Laboratoires, Brussels, BelgiumPurpose: Itraconazole (ITZ) dry powders for inhalation (DPI) composed of nanoparticles (NP) embedded in carrier microparticles were prepared and characterized.Methods: DPIs were initially produced by reducing the ITZ particle size to the nanometer range using high-pressure homogenization with tocopherol polyethylene 1000 succinate (TPGS, 10% w/w ITZ) as a stabilizer. The optimized nanosuspension and the initial microsuspension were then spray-dried with different proportions of or in the absence of mannitol and/or sodium taurocholate. DPI characterization was performed using scanning electron microscopy for morphology, laser diffraction to evaluate the size-reduction process, and the size of the dried NP when reconstituted in aqueous media, impaction studies using a multistage liquid impactor to determine the aerodynamic performance and fine-particle fraction that is theoretically able to reach the lung, and dissolution studies to determine the solubility of ITZ.Results: Scanning electron microscopy micrographs showed that the DPI particles were composed of mannitol microparticles with embedded nano- or micro-ITZ crystals. The formulations prepared from the nanosuspension exhibited good flow properties and better fine-particle fractions, ranging from 46.2% ± 0.5% to 63.2% ± 1.7% compared to the 23.1% ± 0.3% that was observed with the formulation produced from the initial microsuspension. Spray-drying affected the NP size by inducing irreversible aggregation, which was able to be minimized by the addition of mannitol and sodium taurocholate before the drying procedure. The ITZ NP-based DPI considerably increased the ITZ solubility (58 ± 2 increased to 96 ± 1 ng/mL) co

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Purpose: Itraconazole (ITZ) dry powders for inhalation (DPI) composed of nanoparticles (NP) embedded in carrier microparticles were prepared and characterized. Methods: DPIs were initially produced by reducing the ITZ particle size to the nanometer range using high-pressure homogenization with tocopherol polyethylene 1000 succinate (TPGS, 10% w/w ITZ) as a stabilizer. The optimized nanosuspension and the initial microsuspension were then spray-dried with different proportions of or in the absence of mannitol and/or sodium taurocholate. DPI characterization was performed using scanning electron microscopy for morphology, laser diffraction to evaluate the size-reduction process, and the size of the dried NP when reconstituted in aqueous media, impaction studies using a multistage liquid impactor to determine the aerodynamic performance and fine-particle fraction that is theoretically able to reach the lung, and dissolution studies to determine the solubility of ITZ. Results: Scanning electron microscopy micrographs showed that the DPI particles were composed of mannitol microparticles with embedded nano- or micro-ITZ crystals. The formulations prepared from the nanosuspension exhibited good flow properties and better fine-particle fractions, ranging from 46.2% ± 0.5% to 63.2% ± 1.7% compared to the 23.1% ± 0.3% that was observed with the formulation produced from the initial microsuspension. Spray-drying affected the NP size by inducing irreversible aggregation, which was able to be minimized by the addition of mannitol and sodium taurocholate before the drying procedure. The ITZ NP-based DPI considerably increased the ITZ solubility (58 ± 2 increased to 96 ± 1 ng/mL) compared with that of raw ITZ or an ITZ microparticle-based DPI (,10 ng/mL). Conclusion: Embedding ITZ NP in inhalable microparticles is a very effective method to produce DPI formulations with optimal aerodynamic properties and enhanced ITZ solubility. These formulations could be applied to other poorly, SCOPUS: ar.j, info:eu-repo/semantics/published

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Les tumeurs pulmonaires, qu’elles soient primaires (principalement représentées par le cancer du poumon non-à-petites cellules) ou secondaires (métastases), causent la mort de plusieurs centaines de milliers de personnes par an à travers le monde. Malgré les modalités de traitements existantes, un plateau thérapeutique a été atteint avec un taux de survie à 5 ans de maximum 15%. Actuellement, il est connu que le cancer du poumon non-à-petites cellules ainsi que les métastases sont intrinsèquement résistants à l’apoptose.Pour apporter des réponses aux principales problématiques rencontrées avec l’administration systémique de la chimiothérapie conventionnelle qui est principalement constituée d’agents pro-apoptotiques, nous avons développé des formulations à base d’agents antinéoplasiques aux propriétés anticancéreuses non pro-apoptotiques qui sont destinées à être administrées de manière localisée par la voie inhalée. Il faut savoir que l’inhalation est la voie d’administration préférentielle des principales affections respiratoires telles que l’asthme, la bronchopneumonie chronique obstructive et la mucoviscidose. La première partie de ce travail a consisté à produire et à évaluer des formulations à base de témozolomide destinées à être administrées chez la souris porteuse de pseudo-métastases pulmonaires (issues d’un mélanome expérimental, le modèle B16F10), soit via la voie intraveineuse (iv) conventionnelle soit via la voie inhalée à l’aide d’un dispositif endotrachéal approprié. La suspension pour inhalation a été produite à l’aide de technique de réduction de taille et a été stabilisée à l’aide de phospholipides compatibles avec la voie pulmonaire. L’activité anticancéreuse in vitro a été vérifiée pour le témozolomide formulé sous forme de suspension pour inhalation et de solution intraveineuse par rapport à du témozolomide non formulé sur des lignées de cellules cancéreuses de cancer humain NSCLC A549, de glioblastome humain T98G et de mélanome murin B16, Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/nonPublished

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Respiratory disease is the main cause of morbidity and mortality in patients with cystic fibrosis (CF). In particular, patients suffer from chronic infection due to biofilm formation by opportunistic Pseudomonas aeruginosa (32). Therefore, there is an urgent need to develop alternative ways to treat biofilm-associated clinical infections. The aim of this study was to compare the antimicrobial effects in vitro of the combinations tobramycin-clarithromycin and tobramycin-azithromycin against five P. aeruginosa biofilms and to establish the most effective combination. We performed a kinetic study over a period of 28 days of a twice-daily coadministration of the combinations tobramycin-clarithromycin and tobramycin-azithromycin on 12-day-old, mature P. aeruginosa biofilms formed on microplate pegs for 4 clinical isolates and one laboratory strain (PAO1) to simulate the treatment of CF patients with tobramycin inhalation solution (TOBI) through aerosolization. A synergy between tobramycin and clarithromycin was recorded for 3/5 biofilms, with a bacterial decrease of more than 5 log. Conversely, we found an antagonistic activity when 4 μg/ml tobramycin was administered with azithromycin at 2 μg/ml for P. aeruginosa PAO1 and with azithromycin at 2, 20, 50, 100, and 200 μg/ml for P. aeruginosa PYO1. Treatment with tobramycin at 4 μg/ml combined with clarithromycin at 200 μg/ml eradicated all five biofilms, while tobramycin-azithromycin at the same concentrations eradicated only three biofilms. Results of this study suggest that local administration of tobramycin and clarithromycin into the respiratory tract represents a better strategy than the combination tobramycin-azithromycin for the treatment of P. aeruginosa-associated pulmonary infections., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

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