Anne Seidlitz

Determination of permeability coefficients of ophthalmic drugs through different layers of porcine, rabbit and bovine eyes.

To treat ophthalmic diseases like glaucoma or inflammatory disorders topically applied ophthalmic formulations such as eye drops are usually used. In addition, novel ophthalmic implants releasing drug substances locally into different parts of the eye are available today. In the work presented here, the permeability coefficients of selected drugs (ciprofloxacin hydrochloride, lidocaine hydrochloride, timolol maleate) for ophthalmic tissues were determined using side-by-side diffusion chambers (so-called Ussing chambers). Sclera, conjunctiva, cornea, choroidea-retina-complex and a complex of conjunctiva-sclera-choroidea-retina were excised from fresh porcine, rabbit and bovine eyes. In the porcine eye tissues the highest P(app) values were obtained for conjunctiva with the exception of lidocaine. Therefore, it can be estimated that a certain amount of drug diffuses or is transported through conjunctiva after application. The P(app) values for sclera were also higher than those for cornea and even more, the surface area of sclera which is available for drug absorption is much larger than that of cornea when applying an implant. The obtained permeability coefficients for sclera and conjunctiva indicate that the administration of periocular implants can be an alternative to topically applied formulations. The complexes of the tissues were a significantly (p<0.01) stronger barrier to the investigated substances than the separated tissues. Distinct differences in permeability coefficients between the investigated animal tissues were observed. Overall the highest P(app) values for all mounted tissues were obtained with the rabbit, followed by porcine and bovine eyes. Because of these distinct interspecies differences one must be very careful when selecting the proper animal model for the permeability experiments.

Examination of drug release and distribution from drug-eluting stents with a vessel-simulating flow-through cell

The recently introduced vessel-simulating flow-through cell offers new possibilities to examine the release from drug-eluting stents in vitro. In comparison with standard dissolution methods, the additional compartment allows for the examination of distribution processes and creates dissolution conditions which simulate the physiological situation at the site of implantation. It was shown previously that these conditions have a distinct influence on the release rate from the stent coating. In this work, different preparation techniques were developed to examine the spatial distribution within the compartment simulating the vessel wall. These methods allowed for the examination of diffusion depth and the distribution resulting in the innermost layer of the compartment simulating the vessel wall. Furthermore, the in vitro release and distribution examined experimentally were modelled mathematically using finite element (FE) methods to gain further insight into the release and distribution behaviour. The FE modelling employing the experimentally determined diffusion coefficients yielded a good general description of the experimental data. The results of the modelling also provided important indications that inhomogeneous coating layer thicknesses around the strut may result from the coating process which influence release and distribution behaviour. Taken together, the vessel-simulating flow-through cell in combination with FE modelling represents a unique method to analyse drug release and distribution from drug-eluting stents in vitro with particular opportunities regarding the examination of spatial distributions within the vessel-simulating compartment.

Magnesium used in bioabsorbable stents controls smooth muscle cell proliferation and stimulates endothelial cells in vitro.

Magnesium-based bioabsorbable cardiovascular stents have been developed to overcome limitations of permanent metallic stents, such as late stent thrombosis. During stent degradation, endothelial and smooth muscle cells will be exposed to locally high magnesium concentrations with yet unknown physiological consequences. Here, we investigated the effects of elevated magnesium concentrations on human coronary artery endothelial and smooth muscle cell (HCAEC, HCASMC) growth and gene expression. In the course of 24 h after incubation with magnesium chloride solutions (1 or 10 mM) intracellular magnesium level in HCASMC raised from 0.55 ± 0.25 mM (1 mM) to 1.38 ± 0.95 mM (10 mM), while no increase was detected in HCAEC. Accordingly, a DNA microarray-based study identified 69 magnesium regulated transcripts in HCAEC, but 2172 magnesium regulated transcripts in HCASMC. Notably, a significant regulation of various growth factors and extracellular matrix components was observed. In contrast, viability and proliferation of HCAEC were increased at concentrations of up to 25 mM magnesium chloride, while in HCASMC viability and proliferation appeared to be unaffected. Taken together, our data indicate that magnesium halts smooth muscle cell proliferation and stimulates endothelial cell proliferation, which might translate into a beneficial effect in the setting of stent associated vascular injury.

Assessment of different polymers and drug loads for fused deposition modeling of drug loaded implants

&NA; The 3D printing technique of fused deposition modeling® (FDM) has lately come into focus as a potential fabrication technique for pharmaceutical dosage forms and medical devices that allows the preparation of delivery systems with nearly any shape. This is particular promising for implants administered at application sites with a high anatomical variability where an individual shape adaption appears reasonable. In this work different polymers (Eudragit®RS, polycaprolactone (PCL), poly(L‐lactide) (PLLA) and ethyl cellulose (EC)) were evaluated with respect to their suitability for FDM of drug loaded implants and their drug release behaviour was evaluated. The fluorescent dye quinine was used as a model drug to visualize drug distribution in filaments and implants. Quinine loaded filaments were produced by solvent casting and subsequent hot melt extrusion (HME) and model implants were printed as hollow cylinders using a standard FDM printer. Parameters were found at which model implants (hollow cylinders, outer diameter 4–5 mm, height 3 mm) could be produced from all tested polymers. The drug release which was examined by incubation of the printed implants in phosphate buffered saline solution (PBS) pH 7.4 was highly dependent on the used polymer. The fastest relative drug release of approximately 76% in 51 days was observed for PCL and the lowest for Eudragit® RS and EC with less than 5% of quinine release in 78 and 100 days, respectively. For PCL further filaments were prepared with different quinine loads ranging from 2.5% to 25% and thermal analysis proved the presence of a solid dispersion of quinine in the polymer for all tested concentrations. Increasing the drug load also increased the overall percentage of drug released to the medium since nearly the same absolute amount of quinine remained trapped in PCL at the end of drug release studies. This knowledge is valuable for future developments of printed implants with a desired drug release profile that might be controlled by the choice of the polymer and the drug load. Graphical abstract Figure. No caption available. Highlights3D‐printing of quinine loaded implants was established for 4 different polymers.Drug release is highly dependent on the choice of the polymer.Solid quinine dispersions in polycaprolactone formed for all tested drug loadings.

In‐vitro dissolution methods for controlled release parenterals and their applicability to drug‐eluting stent testing

Objectives  Dissolution testing is a powerful tool for the characterization of dosage form performance in vitro under standardized conditions. In spite of the increasing number of parenterally administered medicinal products, currently there are no compendial dissolution test methods designed especially for these types of dosage forms. In addition to classical drug delivery systems, drug/device combination products, such as drug‐eluting stents, are being used increasingly.

In Vitro Dissolution Testing of Drug-Eluting Stents

Drug-eluting stents (DES) have revolutionized the treatment of coronary artery blockage by tremendously reducing the rate of in-stent restenosis and the necessity of repeat revascularization compared to bare-metal stents. They are also gaining increasing importance in other medical fields such as the treatment of certain localized tumors and in glaucoma therapy. DES generally contain most potent drugs, e.g. immunosuppressants or cytostatics, which are supposed to be released in a well controlled manner over time spans which are chosen according to disease progression. Typically, this means that fairly small amounts of drug are released over long periods of time. Therefore, quantification of in vivo plasma levels is often not feasible. Due to this limitation and the fact that tissue levels cannot be determined in humans, in vitro dissolution testing is one of the most powerful tools to gain insight into the release behaviour of DES. This article focuses on the methods for in vitro dissolution testing of DES which are available up to date and highlights the specific characteristics of drug release from stents arising from the composition and the in vivo localization of the dosage form.

In Vitro Determination of Drug Transfer from Drug-Coated Balloons

Drug-coated balloons are medical devices designed to locally deliver drug to diseased segments of the vessel wall. For these dosage forms, drug transfer to the vessel wall needs to be examined in detail, since drug released into the blood is cleared from the site. In order to examine drug transfer, a new in vitro setup was developed combining the estimation of drug loss during advancement to the site of application in a model coronary artery pathway with a hydrogel compartment representing, as a very simplified model, the vessel wall. The transfer of fluorescent model substances as well as the drug paclitaxel from coated balloons to the simulated vessel wall was evaluated using this method. The model was suitable to quantify the fractions transferred to the hydrogel and also to qualitatively assess distribution patterns in the hydrogel film. In the case of fluorescein sodium, rhodamin b and paclitaxel, vast amounts of the coated substance were lost during the simulated passage and only very small fractions of about 1% of the total load were transferred to the gel. This must be attributed to good water solubility of the fluorescent substances and the mechanical instability of the paclitaxel coating. Transfer of the hydrophobic model substance triamterene was however nearly unaffected by the preliminary tracking procedure with transferred fractions ranging from 8% to 14%. Analysis of model substance distribution yielded inhomogeneous distributions indicating that the coating was not evenly distributed on the balloon surface and that a great fraction of the coating liquid did not penetrate the folds of the balloon. This finding is contradictory to the generally accepted assumption of a drug depot inside the folds and emphasizes the necessity to thoroughly characterize in vitro performance of drug-coated balloons to support the very promising clinical data.

Long-term stable hydrogels for biorelevant dissolution testing of drug-eluting stents

Abstract Background: For the purpose of biorelevant dissolution testing hydrogels have recently been used to investigate release and distribution behavior of drugs released from specialized dosage forms such as

Simultaneous magnetic resonance imaging and pharmacokinetic analysis of intramuscular depots

The present pilot study introduces a method that might give novel insights in drug absorption processes from intramuscularly administered depots. An oily suspension or an aqueous solution of paracetamol (6 mg/kg body mass), prednisolone or its hemisuccinate sodium salt for the aqueous solutions (10mg/kg body mass) or diclofenac (10mg/kg body mass) was injected into the muscle tissue of the hind leg of female Lewis-rats (n=47). For the oily suspensions the micronized particles were suspended in medium-chain triglycerides. The aqueous solutions were buffered to a pH of 7.4 ± 0.5. Polyethylene glycol was added as a cosolvent in the formulations containing paracetamol (acetaminophen) and diclofenac and sodium chloride was added to the aqueous solutions of prednisolone hemisuccinate sodium to achieve nearly isotonic formulations. The formed depot was visualized by magnetic resonance imaging (MRI) and characterized with regard to volume and surface area. A 7 T-small animal scanner was used and T1-weighted and T2-weighted sequences including a fat saturation were performed. Simultaneously blood samples were taken and the drugs were quantitatively analyzed. The water based solvent and the oily dispersion agent were visible in the MRI images without the use of contrast agents. Since a free hand injection mostly led to an application directly into the fascia, resulting in a fast removal of the depot, MRI-guided injection was conducted. Comparing pharmacokinetic data with MRI data it was observed that maximal blood levels occurred before the solvent and the dispersion agent were removed from the muscle tissue. Thus, the drug is not absorbed together with the depot. Furthermore, no correlation was found between the shape of the depot and the rate of absorption. Consequently, a higher surface area or volume of the depot did not result in a faster release or absorption of the drugs from the tested formulations. In contrast to the paracetamol and prednisolone formulations the formulations containing diclofenac led to a massive accumulation of interstitial fluid around the injection area being a sign for an acute local reaction. Histological analysis of the muscle tissue revealed a clear correspondence between the amount of interstitial fluid and the extent of infiltrating lymphocytes and granulocytes indicating a tissue response. In conclusion combining MRI with pharmacokinetic data is a suitable method to gain deeper insights into drug absorption processes from intramuscular depots. Furthermore, MRI offers a great possibility detecting local side effects caused by an intramuscularly applied dosage form. This might be very useful in preclinical phases during the development of new intramuscular formulations.

Pharmacokinetics of 1-methyl-L-tryptophan after single and repeated subcutaneous application in a porcine model.

Increased activity of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is associated with immunological and neurological disorders, and inhibition of its enzyme activity could be a therapeutic approach for treatment of these disorders. The aim of the present study was to establish a large animal model to study the accumulation of the potential IDO inhibitor 1-methyltryptophan (1-MT) in blood and different organs of domestic pigs (Sus scrofa domestica). Because 1-MT has not been previously evaluated in pigs, the pharmacokinetics of a single subcutaneous 1-MT application was investigated. Based on this kinetic study, a profile for repeated 1-MT applications over a period of five days was simulated and tested. The results show that a single administration of 1-MT increases its concentrations in blood, with the maximum concentration being obtained at 12 h. Repeated daily injections of 1‑MT generated increasing plasma concentrations followed by a steady-state after two days. Twelve hours after the final application, accumulation of 1-MT was observed in the brain and other organs, with a substantial variability among various tissues. The concentrations of 1-MT measured in plasma and tissues were similar to, or even higher, than those of tryptophan. Our data indicate that repeated subcutaneous injections of 1-MT provide a suitable model for accumulation of 1-MT in plasma and tissues of domestic pigs. These findings provide a basis for further research on the immunoregulatory functions of IDO in a large animal model.