Albin Kristl

Investigation of polymeric nanoparticles as carriers of enalaprilat for oral administration.

Enalaprilat is a typical angiotensin-converting enzyme inhibitor and is very poorly absorbed from the gastrointestinal tract. The aim of this study was to design and characterize poly-(lactide-co-glycolide) (PLGA) and polymethylmethacrylate (PMMA) nanoparticles containing enalaprilat and to evaluate the potential of these colloidal carriers for the transport of drugs through the intestinal mucosa. Nanoparticle dispersions were prepared by the emulsification-diffusion method and characterized according to particle size, zeta potential, entrapment efficiency and physical stability. Effective permeabilities through rat jejunum of enalaprilat in solution and in enalaprilat-loaded nanoparticles were compared using side-by-side diffusion chambers. The solubility of enalaprilat is very low in many acceptable organic solvents, but in benzyl alcohol is sufficient to enable the production of nanoparticles by the emulsification-diffusion process. The diameters of drug-loaded PMMA and PLGA nanoparticles were 297 and 204 nm, respectively. The concentration of the stabilizer polyvinyl alcohol (PVA) in dispersion has an influence on particle size but not on drug entrapment. The type of polymer has a decisive influence on drug content--7 and 13% for PMMA and PLGA nanoparticles, respectively. In vitro release studies show a biphasic release of enalaprilat from nanoparticle dispersions-fast in the first step and very slow in the second. The apparent permeability coefficient across rat jejunum of enalaprilat entrapped in PLGA nanoparticles is not significantly improved compared with enalaprilat in solution.

Polymorphism and pseudopolymorphism: Influencing the dissolution properties of the guanine derivative acyclovir

Abstract In this work we established that acyclovir exists in hydrated form and that the ratio between acyclovir and water molecules in the crystal structure is 3:2. The anhydrous crystalline form of acyclovir was also prepared. Both crystalline forms were examined by means of thermal analyses, X-ray powder diffraction, infrared spectroscopy, solubility and dissolution rate studies. The differences in almost all tested parameters between the acyclovir hydrated and anhydrous forms were observed. They were explained by different crystal forms of the substances examined. It was found, that besides hydrate, two anhydrous forms of acyclovir are present: the unstable one, obtained at a drying temperature below 150°C (which converts to the hydrate almost immediately in the atmosphere), and the stable one, obtained at drying temperatures above 150°C (which shows, on heating to 172°C, the solid-solid transition). It was thus postulated that acyclovir can exist as a pseudopolymorphic and polymorphic solvate.

Mucoadhesive liposomes as new formulation for vaginal delivery of curcumin

Local delivery to the affected area represents the optimal means by which advantageous pharmacological properties of curcumin may be fully exploited as currently, due to the biopharmaceutical limitations associated with this polyphenol, its full beneficial effects remain limited. Curcumin-containing liposomes coated with bioadhesive polymers of natural and synthetic origin (chitosan and Carbopol) were evaluated in vitro. For these purposes, an in vitro model of vaginal mucus was developed allowing the monitoring of curcumin permeability in the conditions mimicking vaginal environment. The model was optimized by varying the amounts of glycoproteins, as compared to the permeabilities determined through isolated bovine mucus. The strength of bioadhesion was evaluated using the isolated bovine mucosa. Both curcumin solution and non-coated curcumin liposomes served as controls. Bioadhesive polymers enabled significantly higher (p<0.05) curcumin permeability through the artificial and isolated bovine mucus compared to the controls. Polymer coating of liposomes resulted in an increase in their bioadhesiveness. Mucoadhesive liposomes can be considered as potential novel drug delivery systems intended for vaginal administration of curcumin.

Simultaneous measurement of imatinib, nilotinib and dasatinib in dried blood spot by ultra high performance liquid chromatography tandem mass spectrometry

Imatinib, dasatinib and nilotinib are three tyrosine kinase inhibitors currently used to treat Bcr-Abl1 positive chronic myelogenous leukaemia (CML). However, achieving maximum benefit with these drugs may require optimal dosing and adherence to therapy. In those cases, therapeutic drug monitoring (TDM) can be a useful tool in managing patients with CML. The paper presents simple and high throughput method for simultaneous determination of all three TKIs in dried blood spot (DBS) samples from CML patients. DBS samples were prepared by applying 10 μL of spiked whole blood onto an Agilent DBS cards. Whole blood spot was punched out of the card, transferred to a well in a 96-well Captiva ND Lipids filter plate. After the addition of isotopically labelled internal standard, the drug was extracted with 0.1% formic acid in methanol. The collected extract (1 μL) was injected onto a Phenomenex Kinetex 50 mm × 2.1 mm C18 column and eluted with acetonitrile gradient into a triple quadrupole ESI-MS/MS Agilent 6460 operated in positive mode. The total run time was only 2.6 min. The method was validated in terms of linearity, selectivity, specificity, accuracy, precision, absolute and relative matrix effect and stability. The effect of haematocrit (Hct) on the accurate concentration determination was also examined. The method was linear in the range of 50-5000 μg/L for imatinib and nilotinib and in the range of 2.5-250 μg/L for dasatinib, with correlation coefficient values higher than 0.997. Lower limits of quantification (LLOQ) were 50 μg/L for imatinib and nilotinib and 2.5 μg/L for dasatinib. The method proved to be accurate (% bias < 13.2) and precise (CV < 10.3%) on intra- as well as on inter-day basis. Sample matrix (% ME=94.5-106.7) and different Hct values had no significant effect on the accuracy of measured concentrations. Samples proved to be stable whilst stored on DBS cards at room temperature or in the refrigerator; however, at 40 °C the stability of dasatinib was compromised. The method presented was successfully applied to clinical samples.

The effect of lipid composition and liposome size on the release properties of liposomes-in-hydrogel

To study the release of liposome-associated drugs into hydrogels, we designed and synthesized two pH-sensitive rhodamine derivatives to use as model compounds of different lipophilicities. The dyes were fluorescent when in the free form released from liposomes into the chitosan hydrogel, but not when incorporated within liposomes. The effect of liposomal composition, surface charge and vesicle size on the release of those incorporated dyes was evaluated. The lipophilicity of the rhodamine derivatives affected both the amount and rate of release. While liposome size had only a minor effect on the release of dyes into the hydrogel, the surface charge affected the release to a greater extent. By optimizing the characteristics of liposomes we could develop a liposomes-in-hydrogel system for application in wound therapy. We further characterized liposomes-in-hydrogel for their rheological properties, textures and moisture handling, as well as their potential to achieve a controlled release of the dye. The polymer-dependent changes in the hydrogel properties were observed upon addition of liposomes. The charged liposomes exhibited stronger effects on the textures of the chitosan hydrogels than the neutral ones. In respect to the ability of the system to handle wound exudates, chitosan-based hydrogels were found to be superior to Carbopol-based hydrogels.

pH and energy dependent transport of ketoprofen across rat jejunum in vitro.

The aim of this study was to elucidate transport mechanisms of ketoprofen (monocarboxylic acid with pK(a) 4.6) across rat jejunum in vitro using side-by-side diffusion cells. When the tissue was incubated on the mucosal and serosal sides with buffer of pH 7.51 (pH of the mucosal surface was 7.08), ketoprofen permeated faster in the mucosal-to-serosal than in the opposite direction. No asymmetry in transport was observed when 2 mM mucus disrupting agent 1,4-dithio-DL-threitol (pH of the mucosal surface increased to 7.21) was added to the mucosal side. Mucosal-to-serosal permeability of ketoprofen increased three times when the pH of the incubation medium was changed from 8.06 (pH of the mucosal surface was 7.34) to 6.07 (pH of the mucosal surface was 5.95), while no pH dependence was found under ATP-depletion caused by sodium azide. In the ketoprofen concentration range from 0.125 to 5 mM no saturation of transport was observed. Moreover, ketoprofen transport was not changed in the presence of 2 mM benzoate, 10 and 20 mM acetate, 20 mM L-lactate (substrates for monocarboxylate transporter 1, MCT1) and 1 mM alpha-cyano-4-hydroxy-cinnamic acid (an inhibitor of MCT1). These results indicate that ketoprofen is transported across rat jejunum in vitro by pH and energy dependent transport mechanisms, and most probably not by MCT1.

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.

A Self-Microemulsifying Drug Delivery System to Overcome Intestinal Resveratrol Toxicity and Presystemic Metabolism

A mixed lipid-mixed surfactant self-microemulsifying drug delivery system (SMEDDS) was developed to exploit the health benefits of resveratrol, a Biopharmaceutical Classification System Class 2 natural polyphenol, subject to extensive intestinal presystemic metabolism. SMEDDS with a mixed lipid phase (castor oil/Capmul MCM 1:1) and a mixed surfactant phase (Kolliphor EL/Kolliphor RH 40 1:1) was developed and evaluated for its self-emulsifying properties and in vitro dispersion. The impact of SMEDDS on the permeability properties of resveratrol and its metabolite fluxes through the rat intestine and Caco-2 cells was monitored. The inhibitory effect of selected SMEDDS components on the efflux transporters multidrug resistance-associated protein and P-gp as well as cytotoxicity was assessed on Caco-2 cells. The formulation allowed for high resveratrol loading (122.5 mg/g SMEDDS), excellent self-emulsifying properties, and very rapid release. When formulated in SMEDDS, resveratrol metabolite efflux significantly declined. The formulation (SMEDDS without incorporated resveratrol) and its individual components did not compromise in vitro cell vitality and integrity. Mixed lipid-mixed surfactant SMEDDS is a prospective formulation to improve resveratrol biopharmaceutical, pharmacokinetic, and toxicological properties, leading the way to resveratrol use not only as a supplement but also as a pharmacological drug.

Polarised transport of monocarboxylic acid type drugs across rat jejunum in vitro: the effect of mucolysis and ATP-depletion.

The transport characteristics of monocarboxylic acid type drugs (ketoprofen, ibuprofen and gemfibrozil) across the excised jejunal segments and artificial (octanol impregnated) membrane in side-by-side diffusion cells were studied. All three model drugs permeated faster across the intestinal tissue in the mucosal-to-serosal direction than in the opposite direction. No polarised transport of tested drugs was observed when the mucosal side of the intestine was treated with mucus disrupting agent, L-cysteine 1% (w/v), which significantly increased the microclimate pH at the mucosal surface of the intestine. Similar effects on the transport characteristics of model drugs and microclimate pH were observed when metabolic inhibitor, sodium azide (10mM), was present in the incubation medium. Furthermore, the direction of proton gradient across the artificial membrane was shown to significantly influence the transport of model drugs across this membrane. The results of this study indicate that the inwardly directed proton gradient maintained by the acidic microclimate pH at the intestinal surface could be considered as a driving force for the transport of monocarboxylic acid type drugs across the intestinal epithelia and could explain rapid absorption of these drugs after oral application.

Preformulation Investigation of the Novel Proton Pump Inhibitor Lansoprazole

Some technologically important physicochemical properties of lansoprazole were investigated. This compound is very unstable, especially in aqueous solutions with low pH. It has one acidic and two basic dissociation constants. Lansoprazole has relatively high solubility in solutions with high pH and is well partitioned from aqueous to n-octanol phase. Under conditions examined in this study, lansoprazole was not hydroscopic and did not decompose at higher relative humidities.