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A series of photoactivatable CO‐releasing molecules (PhotoCORMs) was prepared from manganese pentacarbonyl bromide and 1H‐benzimidazol‐2‐ylmethyl‐(N‐phenyl)amine ligands (L) bearing different electron‐donating and electron‐withdrawing groups R = H, 4‐CH\(_3\), 4‐OCH\(_3\), 4‐Cl, 4‐NO\(_2\), 2‐, 3‐, and 4‐COOCH\(_3\) on the phenyl substituent to give octahedral manganese(I) complexes of the general formula [MnBr(CO)\(_3\)(L)]. Aerated DMSO solutions of the compounds are stable in the dark for 16 h with no CO release. However, the compounds rapidly release CO upon illumination at 412–525 nm, depending on the substitution pattern. Its influence on the photophysical and photochemical properties was systematically explored using UV/Vis spectroscopy and CO release measurements with a commercial gas sensor system. In the nitro‐substituted compound, the electronically excited state switched from benzimidazole‐ to phenyl‐centered, leading to a markedly different photochemical behavior of this visible‐light activated PhotoCORM.
Two chiral chemical molecules being mirror images of each other, also referred to as enantiomers, may have different pharmacokinetic, pharmacodynamic, and toxicological effects. Thus, pharmaceutical manufacturers and authorities are increasingly interested in the approval of enantiopure drugs. However, the isomeric purity and the limits for isomeric impurities have to be specified applying enantioselective analytical methods, such as capillary electrophoresis.
The separation of enantiomers in capillary electrophoresis may be improved by the addition of ionic liquids to the background electrolyte. The aim of this work was to investigate the influence of different separation conditions on the enantioseparation of phenethylamines in background electrolytes containing ionic liquids based on tetrabutylammonium cations.
Best chiral separations were achieved at acidic pH values using phosphate buffers containing 125 mmol/L tetrabutylammonium based salts. Different reasons explaining enhanced enantioseparations in buffers containing ionic liquids were found. First, due to an improvement of the cyclodextrin solubility, the addition of ionic liquids to the background electrolyte enables the use of higher concentrations of these chiral selector. Furthermore, the adsorption of tetrabutylammonium cations to the negatively charged capillary surface results in a reduction of the electroosmotic flow. Hence, the resulting prolongation of migration times leads to a longer period of time for the separation of temporarily formed diastereomeric analyte cyclodextrin complexes, which yields improved enantioseparation. Additionally, due to a decrease of the adsorption of positively charged phenethylamine analyte molecules to capillary surface silanol groups, the adsorption of ionic liquid cations inhibits peak broadening. A further reason explaining an enhanced enantioseparation by the addition of ionic liquids to the background electrolyte is a competition between tetrabutylammonium cations and analyte enantiomers for the inclusion into cyclodextrin cavities.
Furthermore, the influence of different chiral counterions, combined with tetrabutylammonium cations, on the enantioseparation of phenethylamines was investigated. Solely anions based on the basic proteinogenic amino acids L lysine and L arginine yielded chiral separation results superior to those achieved using achiral tetrabutylammonium chloride as background electrolyte additive. Especially the application of tetrabutylammonium L argininate gave very good enantioseparations of all investigated ephedrine derivatives, which might be explained by the ability of L arginine to affect the formation of complexes between analytes and cyclodextrins.
Besides the investigation of the influence of ionic liquids on the enantioseparation, complexes between phenethylamine enantiomers and β cyclodextrin derivatives were characterized by affinity capillary electrophoresis. The binding constants between analyte enantiomers and cyclodextrins and the electrophoretic mobilities of the temporarily formed complexes were determined and compared to the observed chiral resolution values. While neither the calculated binding constants nor their differences correlated with the quality of the enantioseparation, a strong correlation between the differences of the electrophoretic mobilities of the complexes and the chiral resolution values was found.
Upon approval of a drug, the stability of the API and the FPP has to be studied intensively because it determines the shelf-life. If a drug is found to be stable, the expiry date is arbitrary set to five years at the maximum, if a drug tends to undergo degradation, the expiry date is set shorter. The drug product must comply with predefined specifications in accordance with the ICH guidelines Q6A and Q6B during its entire market life. The content of the active substance is required to be within a specification of 95–105% of its labeled claim until expiry corresponding to the ICH guideline Q1A(R2). However, there is little or scattered literature information addressing the stability of drug products beyond their expiry dates. The objective of this thesis was to study and assess the long-term stability of a collection involving numerous pure drug substances and ampoules manufactured in the 20th century. The content and the impurity profile were examined by means of appropriate analytical methods, mainly using liquid chromatography. The results were compared to data being available in the literature. Assessing the stability regarding the dosage form and the affiliation of the drug class was conducted.
The experimental studies comprise the examination of 50 drug substances manufactured 20–30 years ago and 14 long expired ampoules which were older than 40 years in the time of analysis, exceeding many times the maximum shelf life of five years.
For investigation of the solid drug substances, pharmacopoeial methods were applied as far as possible. Indeed, results of the study showed that 44 tested substances still complied with the specification of the Ph. Eur. with regard to the content and impurity profile, even after more than two decades of storage.
For analysis of the injection solutions, HPLC-UV and HPLC-ESI/MS techniques were applied, commonly based on liquid chromatography methods of the Ph. Eur. for determination of related substances. Each method was further validated for its application to ensure accurate API quantification corresponding to ICH Q2(R1). Quite a few ampoules were identified to show surprisingly high stability. In spite of their age of 53–72 years, APIs such as caffeine, etilefrine, synephrine, metamizole sodium, furosemide, and sodium salicylate complied with the specified content that is valid nowadays, respectively. Nevertheless, typical degradation reaction, e.g. hydrolysis, oxidation, or isomerization, was observed in all remaining ampoules. Various degrees of hydrolysis were revealed for scopolamine, procaine, and adenosine triphosphate, the contents were decreased to 71%, 70%, and 15% of the declared concentrations, respectively. In the epinephrine and dipyridamole ampoules, oxidative degradation has been occurred, finding respective API contents of more or less 70%. For dihydroergotamine, excessive decomposition by epimerization was observed, resulting in an API content of 21% and degradation by isomerization was found in lobeline, still containing 64% of the labeled claim.
In conclusion, supported by the data of the present studies and the literature, defining and authorizing a longer shelf-life may be applicable to numerous pharmaceuticals which should be considered by pharmaceutical manufacturers and regulatory authorities, if justified based on stability studies. A general extension of the shelf-lives of drug products and the abolishment or extension of the maximum shelf-life limit of five years would prevent disposing of still potent medications and save a lot of money to the entire health care system.
GPCRs, particularly muscarinic receptors (mAChRs), are significant therapeutic targets in many physiological conditions. The significance of dualsteric hybrids selectively targeting mAChR subtypes is their great advantage in avoiding undesired side effects. This is attained by exploitation of the high affinity of ligand-binding to the orthosteric site and the structural diversity of the allosteric site to target an individual mAChR subtype, as well as offering signal bias to avoiding undesired transduction pathways. Furthermore, dualsteric targeting of mAChR subtypes helps in the elucidation of the physiological role of each individual mAChR subtype.
The first project was the attempt of synthesis of the M2-preferring ligand AFDX-384. AFDX-384 is known to preferentially bind to the M2 receptor subtype as an orthosteric antagonist, with partial interaction with residues in the allosteric site. This project aimed to re-trace the synthesis route of AFDX-384, to open the door to its upscaling and the future synthesis of AFDX-type dualsteric ligands. The multi-step synthesis of AFDX-384 is achieved through the synthesis of its 2 precursors, the chloro acyl derivative VIII and the piperidinyl derivative IV. Upscaled synthesis of the piperidinyl derivative IV was attained. Synthesis of the chloro acyl compound VIII was attempted. Several trials to synthesize the benzopyridodiazepine nucleus as well as its chloro-acylation resulted in the production of the novel crystal structures V and VI. X-ray crystallography was also done for crystallized molecules of the closed-ring benzopyridodiazepine VII that was previously synthesized. Chloro-acylation reactions of compound VII using phosgene seem to be attainable when done using reflux overnight. However, the use of methanol to aid in elution during silica gel column chromatography converted the expected product to the carbamate analogue IX. Hence, further attempts in purification should refrain from the use of methanol. The use of triphosgene instead of phosgene demonstrates a cleaner route for further upscaled synthesis.
The second project was the synthesis of dualsteric ligands involving variable orthosteric and allosteric moieties. Four different types of hybrids have been created over multiple steps. Dualsteric ligands have been synthesized using either a phthalimido- or 1,8-naphthalimidopropylamino moiety as the allosteric-binding group, coupled to either N-desmethyl pirenzepine or N-desmethyl clozapine using variable chain lengths. Furthermore, the synthesis of the dualsteric ligands involving N-desmethyl clozapine linked to either the super-agonist iperoxo or acetylcholine, and being connected using variable alkane chain lengths. Several reaction conditions have been investigated throughout the analysis of the optimal condition to conduct the critical final step of synthesis of these dualsteric hybrids, which involves the linking of the two segments of the hybrid together. The optimal method, which produced the least side products and highest yield, was to connect the two intermediates of the compound in absence of base, catalyst or microwaves while stirring at 35 °C for several days using acetonitrile as solvent (silica gel TLC monitoring, 0.2 M aqueous KNO3/MeOH 2:3). The ideal purification methods for the final compounds were found to be either crystallization from the reaction medium or using C18 reverse phase silica gel flash chromatography (using H2O/MeOH solvent system). All the hybrids will be subjected to pharmacological testing using the appropriate FRET assays.
Polyphenols exert beneficial effects in type 2 diabetes mellitus (T2DM). However, their mechanism of action remains largely unknown. Endothelial Akt-kinase plays a key role in the pathogenesis of cardiovascular complications in T2DM and therefore the modulation of its activity is of interest. This work aimed to characterize effects of structurally different polyphenols on Akt-phosphorylation (pAkt) in endothelial cells (Ea.hy926) and to describe structure-activity features. A comprehensive screening via ELISA quantified the effects of 44 polyphenols (10 µM) on pAkt Ser473. The most pronounced inhibitors were luteolin (44 ± 18%), quercetin (36 ± 8%), urolithin A (35 ± 12%), apigenin, fisetin, and resveratrol; (p < 0.01). The results were confirmed by Western blotting and complemented with corresponding experiments in HUVEC cells. A strong positive and statistically significant correlation between the mean inhibitory effects of the tested polyphenols on both Akt-residues Ser473 and Thr308 (r = 0.9478, p = 0.0003) was determined by immunoblotting. Interestingly, the structural characteristics favoring pAkt inhibition partially differed from structural features enhancing the compounds’ antioxidant activity. The present study is the first to quantitatively compare the influence of polyphenols from nine different structural subclasses on pAkt in endothelial cells. These effects might be advantageous in certain T2DM-complications involving over-activation of the Akt-pathway. The suggested molecular mode of action of polyphenols involving Akt-inhibition contributes to understanding their effects on the cellular level.
The pharmacokinetics in patients with cystic fibrosis (CF) has long been thought to differ considerably from that in healthy volunteers. For highly protein bound β-lactams, profound pharmacokinetic differences were observed between comparatively morbid patients with CF and healthy volunteers. These differences could be explained by body weight and body composition for β-lactams with low protein binding. This study aimed to develop a novel population modeling approach to describe the pharmacokinetic differences between both subject groups by estimating protein binding. Eight patients with CF (lean body mass [LBM]: 39.8 ± 5.4kg) and six healthy volunteers (LBM: 53.1 ± 9.5kg) received 1027.5 mg cefotiam intravenously. Plasma concentrations and amounts in urine were simultaneously modelled. Unscaled total clearance and volume of distribution were 3% smaller in patients with CF compared to those in healthy volunteers. After allometric scaling by LBM to account for body size and composition, the remaining pharmacokinetic differences were explained by estimating the unbound fraction of cefotiam in plasma. The latter was fixed to 50% in male and estimated as 54.5% in female healthy volunteers as well as 56.3% in male and 74.4% in female patients with CF. This novel approach holds promise for characterizing the pharmacokinetics in special patient populations with altered protein binding.
The aim of this thesis was the application of the functional prepolymer NCO-sP(EO-stat-PO) for the development of new biomaterials. First, the influence of the star-shaped polymers on the mechanical properties of biocements and bone adhesives was investigated. 3-armed star-shaped macromers were used as an additive for a mineral bone cement, and the influence on the mechanical properties was studied. Additionally, a previously developed bone adhesive was examined regarding cytocompatibility. The second topic was the examination of novel functionalization steps which were performed on the surface of electrospun fibers modified with NCO-sP(EO-stat-PO). This established method of functionalizing electrospun meshes was advanced regarding the modification with proteins which was then demonstrated in a biological application. Two different kinds of antibodies were immobilized on the fiber surface in a consecutive manner and the influence of these proteins on the cell behavior was investigated. The final topic involved the quantification of surface-bound peptide sequences. By functionalization of the peptides with the UV-reactive molecule 2-mercaptopyridine it was possible to quantify this compound via UV measurements by cleavage of disulfide bridges and indirectly draw conclusions about the number of immobilized peptides.
In the field of mineral biocements and bone adhesives, NCO-sP(EO-stat-PO) was able to influence the setting behavior and mechanical performance of mineral bone cements based on calcium phosphate chemistry. The addition of NCO-sP(EO-stat-PO) resulted in a pseudo-ductile fracture behavior due to the formation of a hydrogel network in the cement, which was then mineralized by nanosized hydroxyapatite crystals following cement setting. Accordingly, a commercially available aluminum silicate cement from civil engineering could be modified.
In addition, it could be shown that the use of NCO-sP(EO-stat-PO) is beneficial for adjusting specific material properties of bone adhesives. Here, the crosslinking behavior of the prepolymer in an aqueous medium was exploited to form an interpenetrating network (IPN) together with a photochemically curing poly(ethylene glycol) dimethacrylate (PEGDMA) matrix. This could be used for the development of a bone adhesive with an improved adhesion to bone in a wet environment. The developed bone adhesive was further investigated in terms of possible influences of the initiator systems. In addition, the material system was tested for cytocompatibility by using different cell lines.
Moreover, the preparation of electrospun fiber meshes via solution electrospinning consisting of poly(lactide-co-glycolide) (PLGA) as a backbone polymer and NCO-sP(EO-stat-PO) as functional additive is an established method for the application of the meshes as a replacement of the native extracellular matrix (ECM). In general, these fibers reveal diameters in the nanometer range, are protein and cell repellent due to the hydrophilic properties of the prepolymer and show a specific biofunctionalization by immobilization of peptide sequences. Here, the isocyanate groups presented on the fiber surface after electrospinning were used to carry out various functionalization steps, while retaining the properties of protein and cell repellency. The modification of the electrospun fibers involved the immobilization of analogs or antagonists of tumor necrosis factor (TNF) and the indirect detection of these by interaction with a light-producing enzyme. Here, a multimodal modification of the fiber surface with RGD to mediate cell adhesion and two different antibodies could be achieved. After culturing the cell line HT1080, the pro- or anti-inflammatory response of cells could be detected by IL-8 specific ELISA measurements.
Furthermore, the quantification of molecules on the surface of electrospun fibers was investigated. It was tested whether the detection by means of super-resolution microscopy would be possible. Therefore, experiments were performed with short amino acid sequences such as RGD for quantification by fluorescence microscopy. Based on earlier results, in which a UV-spectrometrically active molecule was used to detect the quantification of RGD, it was shown that short peptides can also be quantified in a small scale on flat functional substrates (2D) such as NCO-sP(EO-stat-PO) hydrogel coatings, and modified electrospun fibers produced from PLGA and NCO-sP(EO-stat-PO) (3D). In addition, a collagen sequence was used to prove that a successful quantification can be carried out as well for longer peptide chains.
These studies have revealed that NCO-sP(EO-stat-PO) can serve as a functional additive for many applications and should be considered for further studies on the development of novel biomaterials. The rapid crosslinking reaction, the resulting hydrogel formation and the biocompatibility are to be mentioned as positive properties, which makes the prepolymer interesting for future applications.
Molecular Effects of Polyphenols in Experimental Type 2 Diabetes Mellitus and Metabolic Syndrome
(2019)
The growing prevalence of type 2 diabetes mellitus (T2DM) demands novel therapeutic and adjuvant strategies. Polyphenols (PPs) are plant secondary metabolites. Epidemiological studies demonstrate an inverse relationship between their increased intake and the risk of development of T2DM and cardiovascular complications. However, the PPs’ mechanism of action remains largely unknown. The present work aimed to expand knowledge regarding the effects of PPs on diabetes relevant molecular targets.
Pycnogenol® (PYC) is a standardized pine bark extract which consists of oligomeric and monomeric PPs. Its anti-diabetic effects have been demonstrated in clinical trials. As a part of a human study involving 20 healthy volunteers, the extract’s effects on dipeptidyl peptidase IV (DPP IV) were investigated. This protease terminates the insulin secretagogue action of incretins. Its inhibition is a promising strategy in T2DM treatment. This study uncovered that PYC-intake of 100 mg daily over 14 days statistically significantly reduced DPP IV serum concentrations by 8.2 % (n= 38, p= 0.032). Contrary to expectations, this decrease was not paralleled by a reduction in the serum DPP IV enzymatic activity. To the best of our knowledge, the present study was the first investigating the effects of PPs on DPP IV serum concentrations and activities in humans. The finding that PYC is capable of reducing DPP IV serum concentrations might be important with regard to diabetes, where DPP IV levels are increased.
Screenings for PPs’ in vitro effects on DPP IV activity were performed employing a purified enzyme. The effects of tested PPs (among which PYC ingredients) at a physiologically relevant concentration of 5 µM were weak (< 10 %) and too small compared to the reference compound sitagliptin, and thus not likely to be clinically relevant. This result is in discordance with some published data, but consistent with the outcome from the present human study. In addition, fluorescence interactions with the experimental setup were registered: under certain conditions urolithin B exhibited an autofluorescence which might mask eventual inhibitory activity. Quercetin quenched the fluorescence slightly which might contribute to false positive results. No statistically significant effects of selected constituents and metabolites of PYC on the total DPP IV protein expression were observed in 3T3-L1 adipocytes. Thus, the lower DPP IV in vivo concentrations after intake of PYC cannot be explained with down-regulation of the DPP IV expression in adipocytes.
Akt kinase is responsible for the transmission of insulin signals and its dysregulation is related to insulin resistance and plays an important role in development of cardiovascular complications in T2DM. Thus, the modulation of the phosphorylation status of endothelial Akt-kinase, respectively its activity, might be a promising strategy in the management of these pathologies. This work aimed to uncover the effects of PPs from different structural subclasses on Akt-phosphorylation (pAkt) in endothelial cells (Ea.hy926). Short-term effects (5 – 30 min) were investigated at a concentration of 10 µM. In a pilot study two model PPs induced a moderate, but reproducible inhibition of pAkt Ser473 of 52.37 ± 21.01 % (quercetin; p= 0.006, n= 3) and 37.79 ± 7.14 % (resveratrol; p= 0.021, n= 4) compared to the negative control. A primary screening with Western blot analysis investigated the effects of eight compounds from different subclasses on pAkt Ser473 and Thr308 to reveal whether the observed inhibition PPs a group effect or specific to certain compounds. In addition to resveratrol and quercetin, statistically significant inhibitions of pAkt Ser473 were induced by luteolin (29.96 ± 11.06 %, p< 0.01, n= 6) and apigenin (22.57 ± 10.30 %, p< 0.01, n= 6). In contrast, genistein, 3,4,5-trimethoxystilbene, taxifolin and (+)-catechin caused no inhibition. A strong positive and statistically significant correlation between the mean inhibitory effects of the tested PPs on both Akt-residues Ser473 and Thr308 (r= 0.9478, p= 0.0003) was determined. A comprehensive secondary screening via ELISA involving 44 compounds from nine structural groups quantified the effects of PPs on pAkt Ser473 to uncover potential structure-activity features. The most potent inhibitors were luteolin (44.31 ± 17.95 %), quercetin (35.71 ± 8.33 %), urolithin A (35.28 ± 11.80 %), apigenin (31.79 ± 6.16 %), fisetin (28.09 ± 9.09 %), and resveratrol (26.04 ± 5.58 %). These effects were statistically significant (p< 0.01, n= 3 to 6). Further lead structure optimization might be based on the fact that the effects of luteolin and resveratrol also differed statistically significantly from each other (p= 0.008).
To the best of our knowledge, the present study is the first to compare quantitatively the short term effects of PPs from different subclasses on pAkt in endothelial cells. Basic structure-activity relationships revealed that for flavones and flavonols the presence of a C2=C3 double bond (ring C) was essential for inhibitory activity and hydroxylation on the m- and p- positions in the ring B contributed to it. For stilbenoids, three free OH-groups appeared to be optimal. The comparison of the inhibitory potentials of ellagic acid and its microbial metabolites showed that urolithin A was statistically significantly more effective than its progenitor compound. Despite their structural similarities, the only active compound among all urolithins tested was urolithin A, hydroxylated at the C3 and C8 positions. This suggested a specific effect for urolithin A. Based on the common structural determinants and molecular geometry of the most active PPs a pharmacophore model regarding Akt-inhibition was proposed.
In summary, the effects of a wide variety of PPs from diverse structural subclasses on the in vitro phosphorylation of endothelial Akt were quantitatively analyzed for the first time, the effects of previously undescribed compounds were determined and structure activity relationships were elucidated. The inhibitory potential of individual PPs might be beneficial in cases of sustained over-activation of Akt-kinase and its substrates such as S6 kinase as reported for certain T2DM-related pathological states, such as insulin resistance, endothelial dysfunction, excessive angiogenesis, vascular calcification, and insulin triggered DNA-damage. The results of the present work suggest potential molecular mechanisms of action of PP involving Akt-inhibition and DPP IV-down-regulation and thus contribute to the understanding of anti-diabetic effects of these compounds on the molecular level.
The number of active pharmaceutical ingredients (APIs) exhibiting a low solubility in aqueous media or a slow dissolution rate kept rising over the past years urging formulation scientists to explore new ways to tackle poor solubility and to enable oral absorption from such compounds. Bioavailability of poorly water-soluble compounds can be improved by increasing the dissolution rate and/or by increasing the gastro intestinal concentration through transient supersaturation. The dissolution rate of the API can be typically modified by the choice of the physical form, the polymorphic form, the powder surface area, and the local pH, while a transient supersaturation can be extended mainly by nucleation or crystallization inhibiting effects. In the present thesis, three strategies were explored to tailor the dissolution rate, the supersaturation and the hydrotropic solubilization of APIs, weak bases, respectively.
The first part of this thesis followed a bioinspired approach to extend the kinetic solubility of salts and co-crystals. API salts and co-crystals are high energy forms that can generate supersaturated solutions with respect to any more stable form, typically the most stable API form in physiological environment. The transient kinetic stabilization of supersaturated states, also termed “parachute effect”, is considered to improve bioavailability and is one aspect of the formulation that can be tailored. Inspiration from plants, which store high concentrations of aromatic bases in their vacuoles via complexation with polyphenols, sparked the evaluation to use hydroxybenzoic acid derivatives for salt or co-crystal engineering. Imatinib was chosen as the model compound for this investigation as its aromaticity and flat molecular architecture could favor interactions with hydroxybenzoic acid derivatives. One 1:1 Imatinib syringate co-crystal (I-SYA (1:1)) and one 1:2 Imatinib syringate co-crystal salt (I-SYA (1:2)) were obtained. Their dissolution assays in simulated intestinal fluid (SIF; a 50 mM phosphate buffer of pH 6.8) revealed that they formed stable solutions for several hours and days, respectively, in contrast to the marketed Imatinib mesylate salt (approx. 1h). This kinetic stability in solution was linked to the nucleation inhibition of the less soluble Imatinib hydrate by syringic acid (SYA). In solution 1H-NMR studies evidenced the aggregation of Imatinib and SYA. The amphiphilic nature of both Imatinib and SYA is considered to drive their association in solution, additionally, multiple intermolecular interactions such as hydrogen bonds and π-π stacking are likely to contribute. The association in solution enabled a phase of extended supersaturation, i.e., a parachute against desupersaturation, while no negative impact of aggregation on the permeability of both Imatinib and SYA was observed.
A prerequisite to reach supersaturation is a rapid dissolution and release of the API from the formulation. Accordingly, the second and third part of this thesis is focused on the so-called “spring effect” of amorphous solid dispersions (ASDs). The addition of a hydrotropic agent, meaning a molecule that can solubilize poorly water-soluble APIs in aqueous solutions (well-known examples of hydrotropes are benzoic acid and nicotinamide) into an amorphous Ciprofloxacin-polymer matrix led to ternary systems with a significantly faster release and higher concentration of the API in SIF as compared to binary ASDs consisting of Ciprofloxacin (CPX) and polymer only. The stronger spring could be rationalized by an improved wetting of the ASD, or/and by a hydrotropic solubilization effect, although these hypotheses need further investigation. Marked differences in the dissolution profiles of binary ASDs were observed in biorelevant fasted simulated intestinal fluid (FaSSIF; a medium containing Na taurocholate (3 mM) and lecithin (0.75 mM) at pH 6.5) as compared to SIF. In FaSSIF, API release from binary polymeric ASDs was largely improved, and the duration of supersaturation was extended. This suggests that the bile salt Na taurocholate and lecithin present in FaSSIF do improve both dissolution rate and supersaturation of ASDs, the two pillars of ASDs as oral enabling formulations. Indeed, bile salts are endogenous surfactants which, together with phospholipids, play an important role in the wetting, solubilization, and absorption of lipophilic compounds.
The aim of the third part of the present thesis was to study ASDs as formulation principles reducing the strong positive food effect of Compound A. By inclusion of Na taurocholate (NaTC) within the matrix of polymeric ASDs a significant improvement of the dissolution rate and the kinetic solubility in SIF were achieved. Transient supersaturated states of up to four orders of magnitude over the equilibrium solubility were obtained. Two ASDs were selected for further in vivo evaluation in dog. The first was a NaTC/Eudragit E based ASD meant to dissolve and release Compound A in the acidic environment of the stomach, where its solubility is the highest. The second relied on the release of Compound A in the neutral environment of the duodenum and jejunum by using an enterically dissolving polymer, HPMC-P. Releasing the API at the site of its putative absorption was an attempt to control supersaturation levels in the duodenum and to prevent portioning and thus dilution effects during transfer from the stomach. In fasted dogs, exposure from the NaTC/HPMC-P ASD was close to that of the reference Compound A formulation under fed conditions, which suggests an improved dissolution rate and kinetic solubility under fasted conditions (historical data). The exposure from the NaTC/Eudragit E ASD was twice as low as from the NaTC/HPMC-P ASD, and also lower compared to Compound A reference formulation, whereas in vitro the parachute effect of the NaTC/Eudragit E ASD was largely superior to that of the NaTC/HPMC-P ASD. A difference in the extend of the parachute could be related to differences in the thermodynamic activity of dissolved molecules from the two ASDs. Indeed, the high instability of the NaTC/HPMC-P ASD could stem from a high thermodynamic activity driving diffusion through membranes, whereas less instable solutions of NaTC/Eudragit E could indicate solubilization effects which often translate into a lower flux through the biological membrane. Additionally, the pH of the environment where dissolution takes place might be an important factor for absorption, and could also account for the difference in exposure from the two ASDs.
The aim of this thesis was to explore how the intimate environment of weak, poorly soluble bases could be functionalized to improve dissolution rate and kinetic solubility. The investigations highlighted that the performance of enabling oral delivery formulations of weak bases in aqueous media can be enhanced at different levels. At one end initial dissolution rate of ASDs can be tailored by introducing hydrotropes or/and bile salts within the polymeric matrix of ASDs. Bile salts, when combined with appropriate polymers, had also a precipitation inhibition effect enabling the maintenance of supersaturation for a bio-relevant period of time. These results set the ground for further investigations to comprehend specific interactions between bile salts and APIs, and potentially polymers at the molecular level. It will be interesting to explore how such complex systems can be exploited in the formulation design of poorly water-soluble APIs. In addition, it was observed that the duration of supersaturation generated by salts/co-crystals can be extended by the pertinent selection of counterions or coformers. The in vivo relevance of these tunings remains to be evaluated, as translation from closed, in vitro systems to the highly dynamic gastrointestinal environment is not straightforward. A better understanding of the contribution of each kinetic stage (dissolution, supersaturation, and precipitation) and their interplay with physiological factors impacting absorption is essential to facilitate the design of formulations with improved pharmacokinetics.
Detailed insight into the internal structure of drug‐loaded polymeric micelles is scarce, but important for developing optimized delivery systems. We observed that an increase in the curcumin loading of triblock copolymers based on poly(2‐oxazolines) and poly(2‐oxazines) results in poorer dissolution properties. Using solid‐state NMR spectroscopy and complementary tools we propose a loading‐dependent structural model on the molecular level that provides an explanation for these pronounced differences. Changes in the chemical shifts and cross‐peaks in 2D NMR experiments give evidence for the involvement of the hydrophobic polymer block in the curcumin coordination at low loadings, while at higher loadings an increase in the interaction with the hydrophilic polymer blocks is observed. The involvement of the hydrophilic compartment may be critical for ultrahigh‐loaded polymer micelles and can help to rationalize specific polymer modifications to improve the performance of similar drug delivery systems.