Mimicking the dynamic Colonic microbiota in vitro to gain a better understanding on the in vivo metabolism of xenobiotics: Degradation of sulfasalazine.

Due to the potential effects of colonic metabolism, the interest in the composition and action of intestinal microbiota has increased significantly throughout the last 10 years. Recently focus is turning to the development and implementation of in vitro tools closely simulating in vivo colonic metabolic processes suitable for routine use. The aim of the present study is to compare the metabolization of the model drug sulfasalazine utilizing the novel dynamic bioreactor MimiCol and a standard static batch fermenter inoculated with cryopreserved faecal microbiota. Major advantages of the novel bioreactor MimiCol are the smaller media volume which is closer to in vivo conditions, the possibility to perform media changes and the closer simulation of in vivo mixing patterns. The study proved that the MimiCol is able to simulate the dynamic conditions found within the ascending colon. The dynamic conditions within the MimiCol led to an almost 2-fold increase of the metabolization rate constant in comparison to the static batch fermenter. Our study was able to prove that the novel dynamic bioreactor MimiCol is able to closely simulate physiologically relevant conditions.

Tumour-specific delivery of siRNA-coupled superparamagnetic iron oxide nanoparticles, targeted against PLK1, stops progression of pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and is projected to be the second leading cause of cancer-related death by 2030. Despite extensive knowledge and insights into biological properties and genetic aberrations of PDAC, therapeutic options remain temporary and ineffective. One plausible explanation for the futile response to therapy is an insufficient and non-specific delivery of anticancer drugs to the tumour site. DESIGN: Superparamagnetic iron oxide nanoparticles (SPIONs) coupled with siRNA directed against the cell cycle-specific serine-threonine-kinase, Polo-like kinase-1 (siPLK1-StAv-SPIONs), could serve a dual purpose for delivery of siPLK1 to the tumour and for non-invasive assessment of efficiency of delivery in vivo by imaging the tumour response. siPLK1-StAv-SPIONs were designed and synthesised as theranostics to function via a membrane translocation peptide with added advantage of driving endosomal escape for mediating transportation to the cytoplasm (myristoylated polyarginine peptides) as well as a tumour-selective peptide (EPPT1) to increase intracellular delivery and tumour specificity, respectively. RESULTS: A syngeneic orthotopic as well as an endogenous cancer model was treated biweekly with siPLK1-StAv-SPIONs and tumour growth was monitored by small animal MRI. In vitro and in vivo experiments using a syngeneic orthotopic PDAC model as well as the endogenous LSL-KrasG12D, LSL-Trp53R172H, Pdx-1-Cre model revealed significant accumulation of siPLK1-StAv-SPIONs in PDAC, resulting in efficient PLK1 silencing. Tumour-specific silencing of PLK1 halted tumour growth, marked by a decrease in tumour cell proliferation and an increase in apoptosis. CONCLUSIONS: Our data suggest siPLK1-StAv-SPIONs with dual specificity residues for tumour targeting and membrane translocation to represent an exciting opportunity for targeted therapy in patients with PDAC.

Rho kinase inhibition mitigates sunitinib-induced rise in arterial pressure and renal vascular resistance but not increased renal sodium reabsorption.

OBJECTIVES: The therapeutic use of the vascular endothelial growth factor (VEGF) antagonist sunitinib is limited by sunitinib-induced hypertension. The hypotheses were tested that sunitinib increases renal vascular resistance (RVR) and renal Na+ reabsorption, and that Rho kinase (ROCK) inhibition blunts sunitinib-induced hypertension. METHODS: Sunitinib actions on human and rat resistance arteries were investigated by myography. The effects of sunitinib alone or in combination with a ROCK inhibitor on arterial pressure and renal function were investigated in rats by radiotelemetry, renal function and metabolism studies accompanied by biochemical, molecular and histological analyses. RESULTS: Sunitinib blunted agonist-induced vasoconstriction and facilitated endothelium-dependent vasodilation. Within 4 days, sunitinib treatment caused arterial pressure and RVR to rise by 30 mmHg and 5 mmHg × ml × min × g kidney weight, respectively, accompanied by reduced glomerular filtration rate and fractional Na+ excretion with unaffected fractional Li+ excretion. ROCK inhibition blunted sunitinib-induced hypertension and prevented the early rise in RVR, but not the decrease in fractional Na+ excretion, which may explain its modest effect on sunitinib-induced hypertension. CONCLUSION: Our data indicate that early sunitinib-induced hypertension is associated with modest alterations in renal vascular function, but markedly increased renal sodium reabsorption, probably due to direct actions of the VEGF antagonist on the collecting duct, suggesting that VEGF receptors regulate renal Na+ absorption.

Development of a pressure-sensitive glyceryl tristearate capsule filled with a drug-containing hydrogel.

The purpose of this work was to develop a new pressure-sensitive dosage form that breaks and releases its content in a fasted stomach at the predominant pressure at the pylorus. The content of the dosage form should be liquid so that the active pharmaceutical ingredient quickly reaches maximum absorption in the upper small intestine. For this purpose glyceryl tristearate capsules were developed, consisting of an extremely brittle shell, with a crushing behavior that can be controlled by modification of the shell thickness. The capsules were filled with a hydroxyethyl cellulose gel containing paracetamol. Dissolution testing using USP apparatus 2, performed for simulating the resting time in the stomach, did not show any release. Studies using a texture analyser showed a correlation between the glyceryl tristearate filling volume and the necessary force to break the capsule. Physiological conditions in dissolution testing, such as movement, pressure and discontinuous medium contact, were set in a stress test device and showed that the dosage forms did not break and release its pharmaceutical ingredient until a pressure of 300 mbar was applied which served as a threshold limit for physiological pressure occurring during gastric emptying of large solids.

Development of pressure-sensitive dosage forms with a core liquefying at body temperature.

Pressure-sensitive dosage forms have been developed that are intended for pulsatile delivery of drugs to the proximal small intestine. The novel dosage forms are composed of insoluble shell and either a hard fat W32 or polyethylene glycol (PEG) 1000 core that are both liquidizing at body temperature. The release is triggered by predominant pressure waves such as contractions of the pylorus causing rupture of the shell and an immediate emptying of the liquefied filling containing the active ingredient. In consequence immediately after the trigger has been effective the total amount of the drug is intended to be available for absorption in the upper small intestine. Both core types were coated with a cellulose acetate film that creates a pressure-sensitive shell in which mechanical resistance is depending on the coating thickness. Results of the texture analysis confirmed a correlation between the polymer load of the coating and the mechanical resistance. The dissolution test performed under conditions of physiological meaningful mechanical stress showed that the drug release is triggered by pressure waves of ⩾300 mbar which are representing the maximal pressure occurring during the gastric emptying.

Preparation and characterization of magnetizable aerosols.

Magnetizable aerosols can be used for inhalative magnetic drug targeting in order to enhance the drug concentration at a certain target site within the lung. The aim of the present study was to clarify how a typical ferrofluid can be atomized in a reproducible way. The influence of the atomization principle, the concentration of magnetic nanoparticles within the carrier liquid and the addition of commonly used pharmaceutical excipients on the aerosol droplet size were investigated. Iron oxide (magnetite) nanoparticles were synthesized by alkaline precipitation of mixtures of iron(II)- and iron(III)-chloride and coated with citric acid. The resulting ferrofluid was characterized by photon correlation spectroscopy and vibrating sample magnetometry. Two different nebulizers (Pari Boy and eFlow) with different atomization principles were used to generate ferrofluid aerosols. A range of substances that influence the surface tension, viscosity, density or vapor pressure of the ferrofluid were added to investigate their impact on the generated aerosol droplets. The particle size was determined by laser diffraction. A stable ferrofluid with a magnetic core diameter of 10.7 ± 0.45 nm and a hydrodynamic diameter of 124 nm was nebulized by Pari Boy and eFlow. The aerosol droplet size of Pari Boy was approximately 2.5 µm and remained unaffected by the addition of substances that changed the physical properties of the solvent. The droplet size of aerosols generated by eFlow was approximately 5 µm. It was significantly reduced by the addition of Cremophor RH 40, glycerol, polyvinyl pyrrolidone and ethanol.

Direct visualization and identification of biofunctionalized nanoparticles using a magnetic atomic force microscope.

Because of its outstanding ability to image and manipulate single molecules, atomic force microscopy (AFM) established itself as a fundamental technique in nanobiotechnology. (1) We present a new modality that distinguishes single nanoparticles by the surrounding magnetic field gradient. Diamagnetic gold and superparamagnetic iron oxide nanoparticles become discernible under ambient conditions. Images of proteins, magnetolabeled with nanoparticles, demonstrate the first steps toward a magnetic analogue to fluorescence microscopy, which combines nanoscale lateral resolution of AFM with unambiguous detection of magnetic markers.

Magneto-optical relaxation measurements of functionalized nanoparticles as a novel biosensor.

Measurements of magneto-optical relaxation signals of magnetic nanoparticles functionalized with biomolecules are a novel biosensing tool. Upon transmission of a laser beam through a nanoparticle suspension in a pulsed magnetic field, the properties of the laser beam change. This can be detected by optical methods. Biomolecular binding events leading to aggregation of nanoparticles are ascertainable by calculating the relaxation time and from this, the hydrodynamic diameters of the involved particles from the optical signal. Interaction between insulin-like growth factor 1 (IGF-1) and its antibody was utilized for demonstration of the measurement setup applicability as an immunoassay. Furthermore, a formerly developed kinetic model was utilized in order to determine kinetic parameters of the interaction. Beside utilization of the method as an immunoassay it can be applied for the characterization of diverse magnetic nanoparticles regarding their size and size distribution.

Determination of the magneto-optical relaxation of magnetic nanoparticles as a homogeneous immunoassay.

The interaction between human eotaxin (hEotaxin) and its polyclonal antibody anti-human eotaxin (anti-hEotaxin) was investigated by means of a novel liquid-phase immunoassay using the magneto-optical relaxation of ferrofluids. The binding quality as well as kinetic properties of the binding partners was determined using specifically binding magnetic probes. For this purpose, magnetic nanoparticles (MNP; DDM128N, Meito Sangyo, Japan) were initially functionalized with streptavidin. The biotin-nylated antibody was conjugated with streptavidin-MNP applying the streptavidin-biotin binding system. Binding reactions were detected by measuring the relaxation of the optical birefringence signal occurring when a pulsed magnetic field is applied to the ferrofluid. The addition of hEotaxin to anti-hEotaxin conjugated MNP in different amounts yielded an enlargement of the mean relaxation time due to the formation of MNP aggregates. In order to express the observed increase of the particles' effective diameter in terms of elementary kinetic processes between antigen and antibody, a kinetic model was introduced. Here, the binding reactions are described by a process of stepwise polymerization. The obtained results were compared with data received from surface plasmon resonance biosensor analysis, a standard tool for biomolecular interaction analysis.