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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.
Since four decades, high-throughput screenings have been conducted in drug discovery, fuelling the identification of potential new drug candidates. This approach, however, often promotes the detection of compounds with undesired physico-chemical properties like poor aqueous solubility or low membrane permeability. Indeed, dissolution and absorption of a drug are prerequisites for systemic exposure and therapeutic effects. Therefore, innovative strategies to optimize unfavourable performance of new drug candidates are in great demand in order to increase drug concentrations at the site of action whilst simultaneously reducing drug variability.
In chapter I of this research work, hydrophobic ion pairing (HIP) is discussed as a promising strategy to improve the bioavailability of BCS class III compounds, which have high aqueous solubility and low permeability. The review points out the limitations of poorly absorbable drugs and details the approach of pairing these APIs with hydrophobic counterions. Apart from the motivation to tailor physico-chemical, biopharmaceutical and toxicological properties of BCS class III compounds, the hydrophobic ion pairing facilitates their formulation into drug delivery systems. Besides advantageous effects, disadvantages of the ion pair formation, such as the decreased aqueous solubility of the ions pair, are critically outlined. Finally, the review covers an overview of non-invasive administration routes permitted after ion pair formation, including oral/enteral, buccal, nasal, ocular and transdermal drug administration. Overall, the HIP approach offers substantial benefits regarding the bioavailability enhancement of BCS class III compounds.
Chapter II concerns GHQ168 developed by Holzgrabe et al., a BCS class II compound characterized by low aqueous solubility and high permeability. GHQ168 was developed for the treatment of human African trypanosomiasis (HAT), a tropical disease for which novel active compounds are urgently needed. This lead compound was found to be very active against trypanosoma brucei brucei and trypanosoma brucei rhodesiense in cell
culture assays, however, the low aqueous solubility prevented further preclinical development. To target this drawback, two different approaches were selected, including (I) the chemical modification and (II) the spray drying of GHQ168. The newly synthesized set of derivatives as well as the spray dried GHQ168 were subjected to a physico-chemical and microbiological characterization. It turned out that both approaches successfully improved aqueous solubility, however, for the derivatives of GHQ168 at the expense of activity. Furthermore, the pharmacokinetic parameters of GHQ168 and of the most active derivatives, GHQ242 and GHQ243, were evaluated. Elimination half-lives between 1.5 to 3.5 h after intraperitoneal administration and modest to strong serum albumin binding for GHQ243 (45%) and GHQ168 (80%) and very high binding (> 99%) for GHQ242 were detected. The spray dried formulation of GHQ168, as well as GHQ242 and GHQ243 were investigated in two in vivo studies in mice infected with t. b. rhodesiense (STIB900), referred to as (I) stringent model and (II) early-treatment model. In the stringent model (2 applications/day on day 3-6 after infection) the mean survival duration (MSD) of mice treated with spray dried GHQ168 exceeded the MSD of the untreated control group (17 days versus 9 days), a difference that was statistically significant. In contrast, no statistical difference was observed for GHQ242 (14 days) and GHQ243 (12 days). GHQ168 was further assessed in the early-treatment model (2 applications/day on day 1-4 after infection) and again a statistically significant improvement of MSD (32 days (end of observation period) versus 7 days) was observed. Finally, exciting antitrypanosomal efficacy for the spray dried formulation of GHQ168 was demonstrated.
NADPH oxidases (NOX) were found to be the main source of endothelial reactive oxygen species (ROS) formation. Chapter III reports on the formulation studies on triazolopyrimidine derivatives from the VAS library, a set of NADPH oxidase inhibitors. These were developed for the treatment of elevated ROS levels, which contribute to the development of cardiovascular diseases. Although in vitro results from numerous studies indicated promising efficacy and selectivity for the VAS-compounds, the low water solubility impeded the in vivo translation and further preclinical development. For this reason, three derivatives, VAS2870, VAS3947, and VAS4024 were physico-chemically characterized and VAS3947, the most soluble compound, was selected for further formulation studies. These approaches included (I) spray drying, (II) microemulsification and (III) complexation with cyclodextrins in order to develop formulations for oral and parenteral application. Solubility improvement of VAS3947 was successfully demonstrated for all preparations as expressed by supersaturation ratios in comparison to the solubility of the unformulated compound. For seven spray dried formulations, the ratio ranged from 3-9, and the ratio for four microemulsions was 8-19 after 120 min, respectively. The six cyclodextrin formulations achieved the highest supersaturation ratio between 3 and 174 after 20 hours. NMR measurements elucidated the inclusion of VAS3947 within the CD’s cavity as well as the interaction with its outer surface. Ultimately, NOX inhibitors were opened for oral and parenteral administration for the first time.
After successful solubility improvement of VAS3947, further investigations towards in vivo studies were conducted including stability studies with a focus on stability in solution and in plasma as presented in chapter IV. Furthermore, permeability and cytotoxicity assays were performed for the first time. It turned out that VAS3947 was instable in buffer and when exposed to light. Moreover, the compound showed decomposition in the presence of mouse microsomes and in human plasma. The VAS compounds contain an oxazol moiety linked to the triazolopyrimidine skeleton via a thioether. This structural element is responsible for the efficacy of the compound class, however it is susceptible to hydrolysis and to further degradation reactions. Moreover, VAS3947 harmed membrane integrity in the cell permeability assays and cytotoxicity investigations in HEK-293 and HEP-G2 cells revealed IC50 values in the same concentration range as reported for efficacy assays. Summarized, it was demonstrated that substances from the VAS library were no appropriate model compounds for ROS investigations nor suitable candidates for further preclinical development.
Ionische Flüssigkeiten (engl. Ionic Liquids = IL) sind organische Salze mit einem Schmelzpunkt von unter 100 °C und bieten einen interessanten Ansatz um die orale Bioverfügbarkeit von schlecht wasserlöslichen Arzneistoffen zu verbessern.
Aufgrund seiner schlechten Wasserlöslichkeit wurde aus dem Wirkstoff BGG492 der Novartis AG eine Ionische Flüssigkeit (IL) mit dem sterisch anspruchsvollen Gegenion Tetrabutylphosphonium hergestellt. Die IL ist ein amorpher, glasartiger Feststoff mit einem Schmelzpunkt von 57 °C. Die freie Säure (FS), das Kaliumsalz (BGG-K+) und die IL (siehe Abb. 69) wurden in festem Zustand mittels polarisationsmikroskopischen Aufnahmen, Röntgen-Pulverdiffraktometrie, Röntgenkristallstrukturanalysen, Infrarot-Spektroskopie und Festkörper-NMR-Spektroskopie untersucht.
Der ionische Charakter der IL in festem Zustand konnte mittels Bandenverschiebung der deprotonierten Sulfonamidgruppe im IR-Spektrum bestätigt werden. In der Röntgenkristallstrukturanalyse konnte gezeigt werden, dass sich die Moleküle der FS in Schichten anordneten, in denen jedes Molekül mit vier Nachbarmolekülen über Wasserstoffbrücken verbunden war. Das BGG-K+ kristallisierte als Monohydrat. In dieser Kristallstruktur bildeten die Kaliumkationen in der bc-Ebene mit den BGG-Anionen ober- und unterhalb Schichten. Im Gegensatz zu der FS waren keine intermolekularen Wasserstoffbrücken zu beobachten. Die 15N-Festkörper-NMR-Spektren des BGG-K+ und der IL zeigten die gleiche chemische Verschiebung für den unsubstituierten Stickstoffes N-1‘ der Pyrazolgruppe und belegten somit ebenfalls die ionische Struktur der IL im festen Zustand. Die amorphe Struktur der IL wurde mittels Röntgen-Pulverdiffraktometrie und Polarisationsmikroskop bestätigt und eine flüssigkristalline Phase konnte ausgeschlossen werden.
Die IL zeigte im Vergleich zu der FS eine 700-fach schnellere Auflösungsrate J und eine signifikante Verlängerung der Dauer der Übersättigung in wässriger Lösung. Der sprunghafte Anstieg der Kon-zentration in Lösung („spring“) und die Dauer der Übersättigung („parachute“) wurden mittels photometrischen und potentiometrischen Titrationen untersucht. Mit Hilfe der NMR-Spektroskopie konnte der Mechanismus der Übersättigung aufgeklärt werden. Das sterisch anspruchsvolle Gegenion Tetrabutylphosphonium verhinderte die Protonierung der deprotonierten Sulfonamidgruppe von BGG. In Lösung kam es zur Bildung von Aggregaten („Cluster“), in die sich das Gegenion teilweise einlagerte. Nach der Protonierung und der Bildung von Kristallisationskeimen präzipitierte die ungeladenen FS und der metastabile Zustand der Übersättigung („parachute“) brach zusammen.
Um den Einfluss der Struktur des Gegenions auf die Auflösungsrate und die Dauer der Übersättigung zu untersuchen, wurden ca. 40 Phosphonium- und Ammonium-Kationen synthetisiert. Die Schmelzpunkte der Phosphonium- und Ammonium-Salze wurden mittels dynamischer Differenzkalorimetrie (DSC) ermittelt. Für das Phosphonium-Salz P3332OH-Bromid konnte eine enantiotrope Umwandlung der Modifikationen mittels temperaturabhängiger XRPD-Messungen bestätigt werden. Die Zelltoxizitäts-Untersuchungen der Phosphonium- und Ammonium-Salze an humanen Leberzellen (HepG2), Nierenzellen (HEK 293T) und murinen Makro-phagenzellen (J774.1) zeigten, dass mit höherer Lipophilie die Zelltoxizität zunahm. Polare Kationen zeigten keine Zytotoxizität (IC50 > 1000 µM). Die Zelltoxizität der Ammonium-Salze war im direkten Vergleich mit den Phosphonium-Salzen etwas geringer.
Die synthetisierten Phosphonium- und Ammonium-Salze, die als Chloride-, Bromide- und Iodide vorlagen, wurden durch Anionenaustausch in Hydroxide umgewandelt. Die Ionischen Flüssigkeiten wurden in einer Säure-Base-Reaktion mit der freien Säure des BGG-Moleküls und den Hydroxiden hergestellt. Der ionische Charakter konnte mittels Bandenverschiebung der deprotonierten Sulfonamidgruppe im IR-Spektrum bestätigt werden.
Die Substanzen waren amorph (XRPD) und die Glasübergangstemperaturen (DSC) bewegten sich für die Mono-Kationen im Bereich zwischen 40 °C – 97 °C, für Dikationen 81 °C - 124 °C und für Trikationen 124 °C - 148 °C. Damit erfüllten einige Substanzen die Definition einer Ionischen Flüssigkeit nicht (Smp. < 100 °C) und wurden daher als Niedrig-Gitter-Enthalpie-Salze (low lattice enthalpy salt = LLES) bezeichnet. Die ILs und LLES zeigten signifikante Unterschiede in der Auflösungsrate J, der Übersättigungszeit und der Wasserdampfsorption.
In dieser Arbeit konnte gezeigt werden, dass allein durch die Auswahl des Gegenions wichtige Parameter für die orale Bioverfügbarkeit gesteuert werden können. Durch diesen Ansatz war es möglich, aus dem sehr schlecht wasserlöslichen Arzneistoff BGG492 Ionische Flüssigkeiten bzw. LLES herzustellen, die sich drastisch schneller auflösten und teilweise über mehrere Stunden übersättigte Lösungen bildeten. Insgesamt zeigte sich, dass durch eine Zunahme der Polarität des Gegenions eine größere Auflösungsrate J und eine geringere Zelltoxizität erzielt werden konnten. Jedoch verringerte sich dadurch die Dauer der Übersättigung in Lösung und erhöhte die Hygroskopizität der ILs und LLES.
The bile system in vertebrates is an evolutionary conserved endogenous solubilization system for hydrophobic fats and poorly water-soluble vitamins. Bile pours out from the gallbladder through the common bile duct into the duodenum triggered by cholecystokinin. Cholecystokinin is released from enteroendocrine cells after food intake. The small intestine is also the absorption site of many orally administered drugs. Most emerging drug candidates belong to the class of poorly water-soluble drugs (PWSDs). Like hydrophobic vitamins, these PWSDs might as well be solubilized by bile. Therefore, this natural system is of high interest for drug formulation strategies. Simulated intestinal fluids containing bile salts (e.g., taurocholate TC) and phospholipids (e.g., lecithin L) have been widely applied over the last decade to approximate the behavior of PWSDs in the intestine. Solubilization by bile can enhance the oral absorption of PWSDs being at least in part responsible for the positive “food effect”. The dissolution rate of PWSDs can be also enhanced by the presence of bile. Furthermore, some PWSDs profit from supersaturation stabilization by bile salts. Some excipients solubilizing PWSDs seemed to be promising candidates for drug formulation when investigated in vitro without bile. When tested in vivo, these excipients reduced the bioavailability of drugs. However, these observations have been hardly examined on a molecular level and general links between bile interaction in vitro and bioavailability are still missing.
This thesis investigated the interplay of bile, PWSDs, and excipients on a molecular level, providing formulation scientists a blueprint for rational formulation design taking bile/PWSD/excipient/ interaction into account. The first chapter focus on an in silico 1H nuclear magnetic resonance (NMR) spectroscopy-based algorithm for bile/drug interaction prediction. Chapter II to IV report the impact of excipients on bioavailability of PWSDs interacting with bile. At last, we summarized helpful in vitro methods for drug formulation excipient choice harnessing biopharmaceutic solubilization in chapter V.
Chapter I applies 1H NMR studies with bile and drugs on a large scale for quantitative structure-property relationship analysis. 141 drugs were tested in simulated intestinal media by 1H NMR. Drug aryl-proton signal shifts were correlated to in silico calculated molecular 2D descriptors. The probability of a drug interacting with bile was dependent on its polarizability and lipophilicity, whereas interaction with lipids in simulated intestinal media components was dependent on molecular symmetry, lipophilicity, hydrogen bond acceptor capability, and aromaticity. The probability of a drug to interact with bile was predictive for a positive food effect. This algorithm might help in the future to identify a bile and lipid interacting drug a priori.
Chapter II investigates the impact of excipients on bile and free drug fraction. Three different interaction patterns for excipients were observed. The first pattern defined excipients that interacted with bile and irreversibly bound bile. Therefore, the free drug fraction of bile interacting drugs increased. The second pattern categorized excipients that formed new colloidal entities with bile which had a high affinity to bile interacting drugs. These colloids trapped the drug and decreased the free drug fraction. The last excipient pattern described excipients that formed supramolecular structures in coexistence with bile and had no impact on the free drug fraction. These effects were only observed for drugs interacting with bile (Perphenazine and Imatinib). Metoprolol’s free drug fraction, a compound not interacting with bile, was unaffected by bile or bile/excipient interaction. We hypothesized that bile/excipient interactions may reduce the bioavailability of bile interacting drugs.
Chapter III addresses the hypothesis from chapter II. A pharmacokinetic study in rats revealed that the absorption of Perphenazine was reduced by bile interacting excipients due to bile/excipient interaction. The simultaneous administration of excipient patterns I and II did not further reduce or enhance Perphenazine absorption. Conversely, the absorption of Metoprolol was not impacted by excipients. This reinforced the hypothesis, that drugs interacting with bile should not be formulated with excipients also interacting with bile.
Chapter IV further elaborates which in vitro methods using simulated intestinal fluids are predictive for a drug’s pharmacokinetic profile. The PWSD Naporafenib was analyzed in vitro with simulated intestinal fluids and in presence of excipients regarding solubility, supersaturation, and free drug fraction. Naporafenib showed a strong interaction with TC/L from simulated bile. Assays with TC/L, but not without identified one excipient as possibly bioavailability reducing, one as supersaturation destabilizing, and the last as bile not interacting and supersaturation stabilizing excipient. A pharmacokinetic study in beagle dogs outlined and confirmed the in vitro predictions.
The Appendix summarizes in vivo predictive methods as presented in chapter I to IV and rationalizes experimental design paving the way towards a biopharmaceutic excipient screening. The first presented preliminary decision tree is transformed into a step-by-step instruction. The presented decision matrix might serve as a blueprint for processes in early phase drug formulation development.
In summary, this thesis describes how a drug can be defined as bile interacting or non-interacting and gives a guide as well how to rate the impact of excipients on bile. We showed in two in vivo studies that bile/excipient interaction reduced the bioavailability of bile interacting drugs, while bile non-interacting drugs were not affected. We pointed out that the bile solubilization system must be incorporated during drug formulation design. Simulated gastrointestinal fluids offer a well-established platform studying the fate of drugs and excipients in vivo. Therefore, rational implementation of biopharmaceutic drug and excipient screening steers towards efficacy of oral PWSD formulation design.