Towards the next generation of drug administration: evaluation of drug delivery systems in vitro, in vivo, and in clinical settings

Currently, many promising therapeutic compounds suffer from substantial drawbacks such as rapid clearance from circulation, poor bioavailability, or severe toxicity in patients. One option to face these challenges might be the usage of nanoparticles as drug delivery systems. There are different classes of formulations, so called nanomedicines, including drug-protein conjugates, drug-polymer conjugates, liposomes, micelles, and polymersomes. In general, main therapeutic fields for engineered nanomaterials are oncology, cardiovascular medicine, neurology, anti-inflammatory drugs, and anti-infectious treatment. Herein (chapter 2.1), polymersomes prepared of the diblock copolymer PDMS b PMOXA are described. These were synthesised and modified to achieve targeting to the asialoglycoprotein receptor of hepatocytes. Conjugation of asialofetuin to the polymersomes’ surface increased uptake of these polymersomes into hepatocytes, when compared to unmodified polymersomes. Moreover, a model compound was successfully encapsulated into the polymersomes and sustained release was achieved. Biocompatibility of the various polymersomes was assessed in vitro, and zebrafish embryos were utilised as a first vertebrate model to evaluate in vivo toxicity. In conclusion, the targeted drug delivery system was safe and well tolerated in vitro as well as in vivo. Successful drug encapsulation and release make it a promising tool for clinical applications in the field of hepatology. Further comprehensive investigations, especially regarding scalability of production process and in vivo characterisation would be needed before stepping forward to the clinics. In addition to nanoparticles, polymers can also be used to synthesise hydrogels. Hydrogels are crosslinked networks formed by a hydrophilic macromolecular polymer. Their mesh like structure and physicochemical properties allow hydrogels to imbibe large amounts of water and to be used as drug delivery systems. Therefore, hydrogels are of special interest in the fields of drug delivery, biosensing, and tissue engineering. In this work (chapter 2.2), a self-assembling chitosan hydrogel based on chemically modified chitosan (a natural polymer) was prepared. Chitosan is known to be biocompatible, biodegradable, bio-renewable, and non-toxic. In addition, it is tissue-regenerating, haemostatic, and immune-stimulating, making it a very promising hydrogel scaffold for wound dressing. The obtained self-assembling chitosan hydrogel had a porous structure enabling loading it with proteins and releasing them in a sustained manner. Moreover, the hydrogel was biodegradable and could be lyophilised. These characteristics make it a promising scaffold for application of therapeutic proteins in the treatment of chronic wounds. It remains to be elucidated how this chitosan hydrogel loaded with therapeutic proteins would behave in vitro as well as in vivo before clinical applications could be considered. Another application for polymers are buccal films (BFs). BFs can be loaded with drugs and enable oral absorption of these drugs upon placing the BFs into the oral cavity, either sublingual, buccal, or palatal. Besides fast onset of action and reduced first pass metabolism, BFs are easy to be administered, even to vulnerable patient populations such as geriatric or paediatric patients who have difficulties with swallowing liquid or solid oral dosage formulations. This makes BFs – amongst other potential applications – highly valuable for phenotyping purposes, especially for these patient groups. Phenotyping is particularly important for drugs with a narrow therapeutic window that need to be carefully dosed, based on the patient’s individual metabolic capacity. One main metabolising enzyme is cytochrome P450 3A (CYP3A). It is assumed that CYP3A is involved in the metabolism of more than 50% of the marketed drugs. Consequently, such an essential metabolism pathway will be frequently affected by various drug-drug interactions including either inhibition of CYP3A enzymes or increase of CYP3A expression. Enzyme activity can vary up to 400-fold and thus, plasma concentrations of co administered drugs can change tremendously resulting in either reduced drug activity or toxic side effects. To phenotype CYP3A, the benzodiazepine midazolam is a well-accepted probe drug, but suffering from the drawback that patients are sedated during phenotyping with pharmacologically active midazolam doses. Therefore, a microdosing approach for phenotyping was recently developed. One elegant option to formulate and administer microdoses are BFs. Herein (chapter 2.3), a clinical study investigating the usability of a microdosed midazolam BF for phenotyping is described. According to the outcomes, such a film can be considered an interesting novel diagnostic tool in the field of personalised medicine. Before making its step into clinical daily practice, it remains to be evaluated how the microdosed midazolam BF reflects increased or inhibited CP3A activity. Moreover, it would be necessary to test the film again in a greater variety of volunteers (e.g. different age or weight) to be able to generalise the results obtained from the former study. As mentioned above, paediatric patients are a vulnerable population with the need for oral formulations that are easy and safe to administer. A medication’s taste and the ability of children to swallow their medicine may greatly influence the selection of a drug, therefore therapy and prescribing practice. The medication palatability is essential for patient acceptance, therapeutic compliance and successful outcome of a therapy. Not only BFs but also oral disintegrating tablets (ODTs) offer great options for this purpose. To test the palatability of a placebo ODT based on Functionalised Calcium Carbonate, a clinical study with 40 children from 2 to 10 years was conducted, in a setup that considered the specific communication challenges with this target group – obviously children cannot be expected to give quantified feedback on likeability, taste, etc. (chapter 2.4). The tablet was highly accepted by children as well as by their parents. Such palatable, inert carrier ODTs could be formulated to contain a wide range of active pharmaceutical ingredients for oral delivery that are currently unavailable as child friendly formulations or exist only as bad tasting liquids. This includes frequently used and highly relevant drugs, such as antibiotics and steroids. Therefore, the tested ODT or similar child-appropriate solid oral dosage forms could improve world-wide access to high quality medicines and adherence for children in future. In summary, a broad variety of innovative, mostly polymer-based application forms for drug ingredients have been preclinically and clinically evaluated and led to relevant insights on how drug availability could be further optimised

PDMS-b-PMOXA polymersomes for hepatocyte targeting and assessment of toxicity

Nanoparticles, such as polymersomes, can be directed to the hepatic asialoglycoprotein receptor to achieve targeted drug delivery. In this study, we prepared asialofetuin conjugated polymersomes based on the amphiphilic di-block copolymer poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PDMS-b-PMOXA). They had an average diameter of 150nm and formed monodisperse vesicles. Drug encapsulation and sustained release was monitored using the hydrophilic model compound carboxyfluorescein. Asialoglycoprotein receptor specific uptake by HepG2 cells in vitro was energy dependent and could be competitively inhibited by the free targeting ligand. Mechanistic uptake studies revealed intracellular trafficking of asialofetuin conjugated polymersomes from early endosomes and to the lysosomal compartment. Polymersomes showed no toxicity in the MTT assay up to concentrations of 500μg/mL. In addition, acute toxicity and tolerability of our PDMS-b-PMOXA polymersome formulations was assessed in vivo using zebrafish embryos as a vertebrate screening model. In conclusion, a hepatocyte specific drug delivery system was designed, which is safe and biocompatible and which can be used to implement liver-specific targeting strategies

Microdosed midazolam for the determination of cytochrome P450 3A activity: Development and clinical evaluation of a buccal film

Cytochrome P450 3A (CYP3A) isozymes metabolize about 50% of all marketed drugs. Their activity can be modulated up to 400-fold, which has great impact on individual dose requirements for CYP3A substrates. The activity of CYP3A can be monitored using the CYP3A substrate midazolam. To avoid pharmacological midazolam effects during phenotyping, a microdosing approach is preferred. However, the preparation of microdosed dosage forms remains a challenge. Fast dissolving buccal films are therefore proposed to facilitate this task. It was the aim of the present study to clinically evaluate a novel buccal film containing microdoses of midazolam for assessment of CYP3A activity. In a randomized, open-label crossover design, the pharmacokinetics of midazolam and its active hydroxy-metabolite, 1'‑OH‑midazolam, was assessed in 12 healthy volunteers after administration of single microdoses of midazolam (30 μg) as buccal film or buccal solution. The buccal film did rapidly disintegrate, was well tolerated, and no adverse events occurred. The film and the solution showed very similar midazolam plasma concentration-time profiles but were not bioequivalent according to EMA and FDA guidelines. For C; max; , AUC; 0-12h; , and AUC; 0-∞; the geometric mean ratios of film to solution, with their 90% confidence intervals in parentheses, were 1.15 (1.00-1.32), 1.16 (1.04-1.28), and 1.19 (1.08-1.31), respectively. As a proxy for CYP3A activity, molar metabolic ratios of midazolam and 1'‑OH‑midazolam were analyzed over time, which revealed good correlations already 1 h or 2 h after application of the film or the solution, respectively. The tested midazolam buccal film is a convenient dosage form that facilitates administration of a phenotyping probe considerably and may potentially be used in special patient populations such as pediatric patients. Clinical Trials.gov Identifier: NCT03204578