Combining lipid based drug delivery and amorphous solid dispersions for improved oral drug absorption of a poorly water-soluble drug.

Two widely applied enabling drug delivery approaches, self-nanoemulsifying drug delivery systems (SNEDDS) and amorphous solid dispersions (ASD), were combined, with the aim of enhancing physical stability, solubilization and absorption of the model drug ritonavir. Ritonavir was loaded at a concentration above its saturation solubility (Seq) in the SNEDDS (superSNEDDS, 250% of Seq). An ASD of ritonavir with polyvinylpyrrolidone-vinyl acetate copolymers (Kollidon® VA64) was prepared by ball milling. Relevant control formulations, which include conventional SNEDDS (90% of Seq), superSNEDDS with a physical mix of Kollidon® VA64 and ritonavir (superSNEDDS+PM) and an aqueous suspension of ritonavir were used. A pharmacokinetic (PK) study in rats was performed to assess the relative bioavailability of ritonavir after oral administration. This was followed by evaluating the formulations in a novel two-step in vitro lipolysis model simulating rat gastric and intestinal conditions. The addition of a ritonavir containing ASD to superSNEDDS increased the degree of supersaturation from 250% to 275% Seq in the superSNEDDS and the physical stability (absence of drug recrystallization) of the system from 48 h to 1 month under ambient conditions. The PK study in rats displayed significantly higher Cmax and AUC0-7h (3-fold increase) and faster Tmax for superSNEDDS+ASD compared to the conventional SNEDDS whilst containing 3 times less lipid than the latter. Furthermore, superSNEDDS+ASD were able to keep the drug solubilised during in vitro lipolysis to the same degree as the conventional SNEDDS. These findings suggest that dissolving an ASD in a superSNEDDS can contribute to the development of novel oral delivery systems with increased bioavailability for poorly water-soluble drugs.

Controlling the fractal dimension in self-assembly of terpyridine modified insulin by Fe2+ and Eu3+ to direct in vivo effects.

Metal ion-induced self-assembly (SA) of proteins into higher-order structures can provide new, dynamic nano-assemblies. Here, the synthesis and characterization of a human insulin (HI) analog modified at LysB29 with the tridentate chelator 2,2':6',2''-terpyridine (Tpy) is described. SA of this new insulin analog (LysB29Tpy-HI) in the presence of the metal ions Fe2+ and Eu3+ at different concentrations was studied in solution by fluorescence luminescence and CD spectroscopy, dynamic light scattering, and small-angle X-ray scattering, while surface assembly was probed by AFM. Unique oligomerization was observed in solution, as Fe2+ yielded small magenta-colored discrete non-native assemblies, while Eu3+ caused the formation of large fractal assemblies. Binding of both metal ions to Tpy was demonstrated spectroscopically, and emission lifetime experiments revealed a distinct Eu3+ coordination geometry that included two water molecules. SAXS suggested that LysB29Tpy-HI with Fe2+ oligomerized to a discrete, roughly octameric species, while LysB29Tpy-HI with Eu3+ gave very large assemblies that could be modelled as fractals. The fractal dimensionality increased with the Eu3+ concentration. We propose that this is a consequence of Eu3+ binding to both Tpy and to free carboxylic acid groups on the insulin surface. LysB29Tpy-HI maintained insulin receptor affinity, and showed extended blood glucose lowering and plasma concentration after subcutaneous injection in rats. The combination of metal ion directed SA and native SA provides control of nano-scale fractal dimensionality and points towards use in therapeutics.

Visualizing the Journey of Fenofibrate through the Rat Gastrointestinal Tract by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging.

Mapping the spatial distribution of a drug throughout the gastrointestinal tract (GIT) after oral ingestion can provide novel insights into the interaction between the drug, the oral drug delivery system, and the GIT. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a molecular imaging technique that can analyze molecules in the cryosections of tissues, determining their localization with a spatial resolution of 10-100 µm. The overall aim of this study was to use MALDI-MSI to visualize the distribution and spatial location of a model prodrug (fenofibrate) through the rat GIT. Furthermore, the distribution and spatial colocalization of taurocholate and phospholipids in the rat GIT in relation to fenofibrate were investigated. Rats were given a fenofibrate suspension of 10 mg/mL by oral gavage. Blood samples were drawn, and the rats were euthanized at three different time points. The GIT was collected and frozen, and MALDI-MSI was applied on cross sections of the stomach and intestine. Fenofibrate was detected by MALDI-MSI throughout the GIT, which also revealed that fenofibrate was hydrolyzed to the active drug fenofibric acid already in the stomach. Furthermore, the presence of lyso-phosphatidylcholine (lyso-PC) and taurocholate was confirmed in the lumen of the small intestine. MALDI-MSI was shown to be a useful qualitative tool for localizing parent prodrugs and active drugs, with a possibility for gaining insight into not only the location for activation but also the role of endogenous molecules in the process.

In vitro and in vivo comparison of microcontainers and microspheres for oral drug delivery.

Microcontainers, which are microfabricated cylindrical devices with a reservoir function, have shown promise as an oral drug delivery system for small molecular drug compounds. However, they have never been evaluated against a relevant control formulation. In the current study, we prepared microcrystalline cellulose (MCC) microspheres as a control for in vitro and in vivo testing of SU-8 microcontainers as an oral drug delivery system. Both dosage forms were loaded with paracetamol and coated with chitosan or polyethylene glycol (PEG) (12 kDa). These coatings were followed by an additional enteric coating of Eudragit® S100. In addition, a control dosage form was coated with Eudragit® alone. The dosage forms were evaluated in vitro, in a physiologically relevant two-step model simulating rat gastrointestinal fluids, and in vivo by oral administration to rats. In vitro, the microcontainers coated with PEG/Eudragit® resulted in a prolonged release of paracetamol compared to the respective microspheres, which was consistent with in vivo observations of a later time (Tmax) for maximum plasma concentration (Cmax) for the microcontainers. For microspheres and microcontainers coated with chitosan/Eudragit®, the time for complete in vitro release of paracetamol was very similar, due to an earlier release from the microcontainers. This trend was supported by very similar Tmax values in vivo. The in vitro in vivo relation was confirmed by a linear regression with R2 = 0.9, when Tmax for each dosage form was plotted as a function of time for 90% paracetamol release in vitro. From the in vivo study, the average plasma concentration of paracetamol 120 min after dosing was significantly higher for microcontainers than for microspheres (0.3 ± 0.1 µg/mL and 0.1 ± <0.1 µg/mL, respectively) - regardless of the coating applied.

Effect of supersaturation on absorption of indomethacin and tadalafil in a single pass intestinal perfusion rat model, in the absence and presence of a precipitation inhibitor.

The effect of the degree of supersaturation (DS) on absorption of the model drugs indomethacin and tadalafil was elucidated in a single-pass intestinal perfusion (SPIP) model in rats. In addition, the performance of the precipitation inhibitor (PI) hydroxypropylmethylcellulose (HPMC) was evaluated when added at a concentration of 0.1% (w/v) to fasted state simulated intestinal fluid (FaSSIF and FaSSIFHPMC) used as perfusion medium. A supersaturated state was created by a solvent shift method where indomethacin or tadalafil dissolved in dimethyl sulfoxide (DMSO) were administered to a segment of the small intestine, which subsequently was perfused with FaSSIF or FaSSIFHPMC. The perfusate was collected for 60 min, and for one group of rats dosed with 30 mg tadalafil, for 120 min. Blood samples were drawn every 15 min. The solubility of indomethacin and tadalafil in the perfusate was determined. The DS of each drug in the perfusate was calculated by dividing the concentration in the perfusate at selected time points with the solubility. The DS was above one for all timepoints for both drugs, thus showing supersaturation during the time of perfusion. For indomethacin, no improvement of the DS was seen when perfusing with FaSSIFHPMC, compared to FaSSIF. For tadalafil, a higher DS was achieved when perfusing with FaSSIFHPMC compared to FaSSIF. Perfusing the drugs with FaSSIFHPMC resulted in a significantly lower area under the curve (AUC0-60 min) for plasma concentrations of indomethacin, and no increase in the AUC0-60 min of plasma concentrations of tadalafil compared to perfusion with FaSSIF. The importance of simultaneously estimating the intraluminal DS and absorption of a drug was demonstrated by the SPIP model in the present study. Further, the study highlights the discrepancy between optimal in vitro supersaturation, intraluminal supersaturation and in vivo performance of two poorly soluble drugs, and further emphasizes the importance of optimization of in vitro methods in order to predict in vivo supersaturation and precipitation of drugs.

Using in vitro lipolysis and SPECT/CT in vivo imaging to understand oral absorption of fenofibrate from lipid-based drug delivery systems.

Using lipid-based drug delivery systems (LbDDS) is an efficient strategy to enhance the low oral bioavailability of poorly water-soluble drugs. Here the oral absorption of fenofibrate (FF) from LbDDS in rats was investigated in pharmacokinetic, in vitro lipolysis, and SPECT/CT in vivo imaging studies. The investigated formulations were soybean oil solution (SBO), a mixture of soybean oil and monoacyl phosphatidylcholine (MAPC) (SBO-MAPC), self-nanoemulsifying drug delivery systems with and without MAPC (SNEDDS-MAPC and SNEDDS, respectively), and an aqueous suspension (SUSP) as a reference. Oral bioavailability of the LbDDS ranged from 27 to 35%. A two-step in vitro lipolysis model simulating rat gastro-intestinal digestion provided in vitro FF solubilisation data to understand oral absorption. During the in vitro lipolysis, most FF was undissolved for SUSP and distributed into the poorly dispersed oil phase for SBO. For the SNEDDS without MAPC, practically all FF solubilised into the aqueous phase during the dispersion and digestion. Adding MAPC to SBO enhanced the dispersion of the oil phase into the digestion media while adding MAPC to SNEDDS resulted in a distribution of 29% of FF into the oil phase at the beginning of in vitro lipolysis. FF distribution into both oil and aqueous phases explained the higher and prolonged oral absorption of LbDDS containing MAPC. To elucidate the relatively low bioavailability of all formulations, FF and triolein were labeled with 123I and 125I, respectively, to study the biodistribution of drug and lipid excipients in a dual isotope SPECT/CT in vivo imaging study. The concentration of radiolabeled drug as a function of time in the heart correlated to the plasma curves. A significant amount of radiolabeled drug and lipids (i.e., 28-59% and 24-60% of radiolabeled drug and lipids, respectively) was observed in the stomach at 24 h post administration, which can be linked to the low bioavailability of the formulations. The current study for the first time combined in vitro lipolysis and dual isotope in vivo imaging to find the root cause of different fenofibrate absorption profiles from LbDDS and an aqueous suspension.

Ex vivo intestinal perfusion model for investigating mucoadhesion of microcontainers.

Micro fabricated delivery systems have shown promise in increasing oral bioavailability of drugs. Micrometer-sized polymeric devices (microcontainers) have the potential to facilitate unidirectional drug release directly into the intestinal mucosa whereby, drug absorption can be enhanced. The aim of this study was to develop an ex vivo model to investigate mucosal adhesion and orientation of microcontainers. Furthermore, to investigate how microcontainers with varying height, shape and material behave in regards to mucoadhesion and orientation. Microcontainers were placed at the top of an inclined piece of porcine small intestine. The tissue was perfused with biorelevant medium followed by microscopic examination to observe the orientation and amount of microcontainers on the tissue. The mucoadhesion of the microcontainers were evaluated based on the observed position on the tissue after being exposed to flow. When comparing the varying types of microcontainers, good adhesion was in general observed since most of the microcontainers were located in the beginning of the intestine. Microcontainers fabricated from the epoxy-based photoresist SU-8 had a slightly better adherence than those fabricated from poly-ɛ-caprolactone (PCL). The orientation of the microcontainers appeare to be dictated mainly by the height. In general, the model showed promising results in evaluating mucoadhesion and orientation.

Biodegradable microcontainers - towards real life applications of microfabricated systems for oral drug delivery.

Microfabrication techniques have been applied to develop micron-scale devices for oral drug delivery with a high degree of control over size, shape and material composition. Recently, microcontainers have been introduced as a novel approach to obtain unidirectional release to avoid luminal drug loss, enhance drug permeation, protect drug payload from the harsh environment of the stomach, and explore the ability for targeted drug delivery. However, in order to eventually pave the way for real life applications of these microfabricated drug delivery systems, it is necessary to fabricate them in biodegradable materials approved for similar applications and with strategies that potentially allow for large scale production. In this study, we for the first time evaluate biodegradable microcontainers for oral drug delivery. Asymmetric poly-ε-caprolactone (PCL) microcontainers with a diameter of 300 µm and a volume of 2.7 nL are fabricated with a novel single-step fabrication process. The microcontainers are loaded with the model drug paracetamol and coated with an enteric pH-sensitive Eudragit® S100 coating to protect the drug until it reaches the desired location in the small intestine. In vitro dissolution studies are performed to assess the drug load and release profile of the PCL microcontainers. Finally, in vivo studies in rats showed a higher bioavailability compared to conventional dosage forms and confirm the potential of biodegradable microcontainers for oral drug delivery.

Developing a predictive in vitro dissolution model based on gastrointestinal fluid characterisation in rats.

The influence of physiological factors on the solubility of drug compounds has been thoroughly investigated in humans. However, as these factors vary between species and since many in vivo studies are carried out in rats or mice, it has been difficult to establish sufficient in vitro in vivo relations. The aim of this study was to develop a physiologically relevant in vitro dissolution model simulating the gastrointestinal (GI) fluids of fasted rats and compare it to previously published in vitro and in vivo data. To develop the in vitro model, the pH was measured in situ in six segments of the GI tract of anesthetised rats, then the fluids from the stomach, the proximal and the distal small intestine were collected and characterized with regard to osmolality, and bile acid and phospholipid concentration. The pH and osmolality were found to increase throughout the GI tract. The bile acids and phospholipids were present in high concentrations in the proximal small intestine, and the bile acid concentration doubled in the distal part, where the phospholipid concentration decreased. Matrix-assisted laser desorption ionisation mass spectrometry imaging was applied on a cross section of the small intestine, to study which bile acids and phospholipid classes were present in the small intestine of rats. Both cholic acid, taurocholic acid and glycocholic acid were detected, and phosphatidylcholine (34:2) was found to be mainly present in the intestinal wall or mucus, whereas lysophosphatidylcholine (16:0) was also detected in the lumen. Based on these observations, biorelevant media were developed to simulate fluids in the stomach and the proximal part of the small intestine in fasted rats. The media were implemented in a two-step in vitro dissolution model, which was found to better predict the in vivo performance of furosemide, when compared to previously published in vitro and in vivo data.

From concept to in vivo testing: Microcontainers for oral drug delivery.

This work explores the potential of polymeric micrometer sized devices (microcontainers) as oral drug delivery systems (DDS). Arrays of detachable microcontainers (D-MCs) were fabricated on a sacrificial layer to improve the handling and facilitate the collection of individual D-MCs. A model drug, ketoprofen, was loaded into the microcontainers using supercritical CO2 impregnation, followed by deposition of an enteric coating to protect the drug from the harsh gastric environment and to provide a fast release in the intestine. In vitro, in vivo and ex vivo studies were performed to assess the viability of the D-MCs as oral DDS. D-MCs improved the relative oral bioavailability by 180% within 4h, and increased the absorption rate by 2.4 times compared to the control. This work represents a significant step forward in the translation of these devices from laboratory to clinic.

Thromboelastography in Selected Avian Species.

Currently available assay methods and reagents are not optimized for evaluating avian hemostasis; therefore, assessing avian coagulopathies is challenging. Recently, thromboelastography (TEG), which measures the viscoelastic properties of blood, has been used clinically in mammalian species to diagnose and characterize hemostatic disorders. To evaluate TEG in healthy individuals of 6 avian species, we modified existing mammalian TEG protocols to allow analysis of citrated, avian whole-blood samples collected from scarlet ibis (Eudocimus ruber) (n = 13), American flamingos ( Phoenicopterus ruber ) (n = 13), helmeted Guinea fowl ( Numida meleagris ) (n = 12), Amazon parrots (Amazona species) (n = 9), Humboldt penguins ( Spheniscus humboldti ) (n = 6), and domestic chickens (n = 16). Activated partial thromboplastin time, prothrombin time, and fibrinogen were measured as a means of comparison. Regardless of the mode of activation, clot formation in the species studied was markedly delayed compared with mammals. Because of prolonged reaction time (14.7-52.7 minutes) with kaolin and diluted tissue factor, undiluted human tissue factor was used in all avian samples because it provided the shortest reaction time. Species differed significantly in reaction time (P = .007), clotting rate (P < .001), rate of clot formation (α angle; P < .001), and maximum amplitude (P < .001) values, indicating that species-specific reference intervals are necessary. Based on these results, TEG with specific reference intervals could prove useful in evaluating avian hemostatic disorders.