Simon Žakelj

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.

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.

In vitro interactions between aged garlic extract and drugs used for the treatment of cardiovascular and diabetic patients

BackgroundDisease preventing effects gained by garlic consumption have been recognized since early period of history, making commercially available garlic supplements attractive to the general public. Possible pharmacokinetic interactions which could occur between applied drugs and aged garlic extract (AGE) are unknown.AimTo test in vitro impact of some garlic phytochemicals on P-glycoprotein (Pgp), the most recognized efflux transporter, and the effect of AGE on passive membrane permeability, absorptive and secretory intestinal transporters.MethodsRat small intestine and Caco-2 cell monolayers, mounted in side-by-side diffusion chambers were used.ResultsHydrophilic sulphur compounds increased Pgp mediated Rhodamine 123 (Rho123) efflux, whereas the lipophilic ones increased Pgp efflux through rat ileum but not through Caco-2 cell monolayers. Increased activities of secretory (Pgp, multidrug-resistance associated protein 2) and absorptive (monocarboxylate transporter 1, organic anion transporting polypeptide) transporters involved in drug absorption were observed in rat small intestine and Caco-2 cell monolayers in the presence of AGE. Transport of drugs mediated by breast cancer resistance protein and H+-oligopeptide transporter 1 was activated in rat intestine but inhibited through Caco-2 cells. Passive membrane permeability of tested compounds remained unaltered through rat small intestine, while significant changes were observed with Caco-2 cell monolayers.ConclusionsDue to the observed in vitro pharmacokinetic interactions between AGE and investigated cardiovascular, antidiabetic and antiviral drugs, in vivo absorption changes are possible, but the magnitude of change depends on the most profound process involved (influx, efflux, passive diffusion) in compounds permeability.

Development of an in-vivo active reversible butyrylcholinesterase inhibitor.

Alzheimer’s disease (AD) is characterized by severe basal forebrain cholinergic deficit, which results in progressive and chronic deterioration of memory and cognitive functions. Similar to acetylcholinesterase, butyrylcholinesterase (BChE) contributes to the termination of cholinergic neurotransmission. Its enzymatic activity increases with the disease progression, thus classifying BChE as a viable therapeutic target in advanced AD. Potent, selective and reversible human BChE inhibitors were developed. The solved crystal structure of human BChE in complex with the most potent inhibitor reveals its binding mode and provides the molecular basis of its low nanomolar potency. Additionally, this compound is noncytotoxic and has neuroprotective properties. Furthermore, this inhibitor moderately crosses the blood-brain barrier and improves memory, cognitive functions and learning abilities of mice in a model of the cholinergic deficit that characterizes AD, without producing acute cholinergic adverse effects. Our study provides an advanced lead compound for developing drugs for alleviating symptoms caused by cholinergic hypofunction in advanced AD.

Decreasing acidity in a series of aldose reductase inhibitors: 2-Fluoro-4-(1H-pyrrol-1-yl)phenol as a scaffold for improved membrane permeation

Targeting long-term diabetic complications, as well as inflammatory pathologies, aldose reductase inhibitors (ARIs) have been gaining attention over the years. In the present work, in order to address the poor membrane permeation of previously reported ARIs, derivatives of N-phenylpyrrole, bearing groups with putative pKa≥7.4, were synthesized and evaluated for aldose reductase inhibitory activity. The 2-fluorophenol group proved the most promising moiety, and further modifications were explored. The most active compound (31), identified as a submicromolar inhibitor (IC50=0.443μM), was also selective against the homologous enzyme aldehyde reductase. Cross-docking revealed that 31 displays a peculiar interaction network that may be responsible for high affinity. Physicochemical profiling of 31 showed a pKa of 7.64, rendering it less than 50% ionized in the physiological pH range, with potentially favorable membrane permeation. The latter was supported from the successful inhibition of sorbitol formation in rat lenses and the ability to permeate rat jejunum.

Influence of luminal monosaccharides on secretion of glutathione conjugates from rat small intestine in vitro

Intestinal efflux transporters can significantly reduce the absorption of the drug after peroral application. In this work we studied secretion of glutathione conjugates triggered by glucose at the luminal side of the intestine. Glucose stimulated secretion of DNPSG, NEMSG and CDNB. We used some different monosaccharides and determined that glucose, galactose and alpha-methylglucopyranoside trigger the secretion, while mannitol and fructose do not. We concluded that interaction with SGLT transporter is the key process necessary for this triggering. To determine which of possible glutathione conjugate transporters (MRP2, MRP4, BCRP or RLIP76) is responsible for the secretion of glutathione conjugates, we used benzbromarone, a MRP inhibitor, and sulfanitran and furosemide, two allosteric MRP2 activators. Benzbromarone inhibited glucose stimulated DNPSG secretion, while allosteric activators additionally increased the secretion. We concluded that MRP2 transporter is related to glucose stimulated DNPSG secretion. Regarding the work of Kubitz et al. we tested the effect of changed medium osmolarity on DNPSG transport and determined that hypoosmolar conditions trigger secretion of DNPSG. These findings suggest that intestinal MRP2 activity has no basal level, but can be stimulated by hypoosmolarity and SGLT transport.

Monitoring of imatinib targeted delivery in human leukocytes.

The success of imatinib therapy in chronic myeloid leukemia is highly influenced by its active transport into target cells. However, the methodology for analytical evaluation of intracellular drug concentration is rare and usually reliant upon the use of radioactively labeled drugs. More specifically, there is no published method available in the literature for the determination of imatinib concentration in granulocytes. To gain further insight into the intracellular drug uptake a very reliable two-stage sample concentration procedure was devised and coupled with a sensitive ultra-high performance liquid chromatography tandem mass spectrometry. The reliability of this sample preparation and sensitivity of the analysis was confirmed by a successful validation of all necessary method parameters to an impressive lower limit of quantification of 0.5 ng imatinib per 10(6) cells still at the signal to noise ratio of 670. The usefulness of the method is further improved with only 6 mL of blood being necessary for patient analysis. The method has been applied to blood samples of 13 CML patients treated with imatinib and all the measured intracellular drug concentrations were within the validated range. These and further measurements will enable the research of factors which may, besides blood plasma concentration, influence the individuals response to imatinib therapy. Furthermore, individualisation of dosing based on the directly measured targeted drug delivery could be possible.

Identification and characterization of the novel reversible and selective cathepsin X inhibitors

Cathepsin X is a cysteine peptidase involved in the progression of cancer and neurodegenerative diseases. Targeting this enzyme with selective inhibitors opens a new possibility for intervention in several therapeutic areas. In this study triazole-based reversible and selective inhibitors of cathepsin X have been identified. Their selectivity and binding is enhanced when the 2,3-dihydrobenzo[b][1,4]dioxine moiety is present as the R1 substituent. Of a series of selected triazole-benzodioxine derivatives, compound 22 is the most potent inhibitor of cathepsin X carboxypeptidase activity (Ki = 2.45 ± 0.05 μM) with at least 100-fold greater selectivity in comparison to cathepsin B or other related cysteine peptidases. Compound 22 is not cytotoxic to prostate cancer cells PC-3 or pheochromocytoma PC-12 cells at concentrations up to 10 μM. It significantly inhibits the migration of tumor cells and increases the outgrowth of neurites, both processes being under the control of cathepsin X carboxypeptidase activity. Compound 22 and other characterized triazole-based inhibitors thus possess a great potential for further development resulting in several in vivo applications.

Absorption and elimination of imatinib through the rat intestine in vitro.

Imatinib is a potent selective inhibitor of tyrosine kinases and is used primarily in the treatment of chronic myeloid leukemia and the gastrointestinal stromal tumour. Although, it is well established that imatinib is a substrate of several transport proteins which are also active in the intestinal mucosa, the mechanisms of imatinib intestinal absorption and elimination were not systematically investigated yet. To do that, we used a Sweetana-Grass type of diffusion chambers with segments of rat intestine as a model of the intestinal mucosa, measured the permeability coefficients of imatinib and its major metabolite (N-desmethyl imatinib) in both directions with and without specific and general inhibition of active transport, and calculated the efflux ratios. The results show that the good bioavailability of imatinib is highly likely achieved by its active absorption from the intestine and that its active elimination through the intestinal mucosa is mediated by a synergistic activity of organic cation transporter 1 in the basolateral membrane and the added activity of two efflux proteins (P-glycoprotein and breast cancer resistant protein) in the apical membrane of enterocytes of the rat ileum. Interestingly, it was found that N-desmethyl imatinib is only transported by P-glycoprotein.

Thematic issue on drug delivery for specific populations.

http://dx.doi.org/10.1016/j.ejps.2015.04.019 0928-0987/ 2015 Elsevier B.V. All rights reserved. The 10th Central European Symposium on Pharmaceutical Technology (CESPT 2014) with the main theme ‘‘Translation of basic concepts into drug delivery for specific populations’’ took place in Portorož, Slovenia in September 18–20, 2014. This scientific event was organized by the Slovenian Pharmaceutical Society and the Faculty of Pharmacy, University of Ljubljana with additional support from fourteen European pharmaceutical associations including EUFEPS. Leading researchers presented a total of 130 contributions showing the present and future advances in providing medication to the specific patient while understanding all the way ‘‘from-lab-to-bed’’ that specific patients have specific needs and that one size no longer fits all in modern pharmacotherapy. Most directly, the new challenges in drug formulation for individual patients were addressed in a session ‘‘Advanced drug delivery systems for specific populations’’. The testing and approaches to the analysis of novel dosage forms for which the traditional pharmacopoeial techniques are inadequate and which the existing guidelines provided by regulatory agencies still do not cover well enough, were discussed under ‘‘Frontiers of biopharmaceutical evaluation to promote early drug registration’’. ‘‘Tuning physicochemical properties of nanomaterials to efficacy and safety’’ was about actual implementation of nanotechnology in the present and future patient oriented medications while some ultimate ‘‘in silico’’ approaches to the individualization of therapy as well as in the optimization of drug design and manufacture were discussed under ‘‘Recent achievements of modeling approach to personalized medicine’’. The editors of the present thematic issue have carefully selected and invited several presenting authors to contribute their views and knowledge in review articles and their cutting edge achievements in research. The symposium as well as the fine contributions to this special issue show that serving the needs of individual patients and groups of patients with specific characteristics is truly becoming an important factor of modern drug development. We can observe that easy to swallow formulations like orodispersible tablets, taste masking approaches, individual dosing enabling formulations like minitablets or printable drugs and formulation approaches which improve the bioavailability or release profiles of medications needed by special populations are an ever more popular topic of academic and industrial research. This does not in any way mean that the traditional challenges of pharmaceutical formulation are becoming obsolete. On the contrary, addressing the issues posed by inconvenient physicochemical properties of drugs such as low solubility or pharmacokinetic characteristics which require modified drug release remains at the centre of attention during formulation development even as patient oriented mentality is ever more incorporated in the drug design. We were also reminded that the needs of individual patients for the ‘‘best fit’’ treatment are not easily defined and that solutions will need to be sought in the simplification of therapeutic drug monitoring as well as ‘‘in silico’’ modelling approaches. Several authors remind us that the ‘‘in silico’’ modelling and predictions in correlation with ‘‘in vitro’’ and ‘‘in vivo’’ drug testing are making their way to the core of processes involved in drug development, manufacturing and biopharmaceutical evaluation as necessary tools to provide the future quality of medication. Overall, the contents of this issue show the solutions of many challenges in designing drug delivery systems for specific populations. We are aware that many questions still remain unanswered and hope that unsolved problems will lead to further, even more intensive research. We wish to thank Prof. M. Brandl and Elsevier for providing us the opportunity for editorship of this special issue and to expand the ideas of CESPT 2014 to a wider audience in the field of pharmaceutical sciences. Finally, we would like to acknowledge the valuable contributions from all the authors of the selected papers as well as other delegates at CESPT 2014 for inspiring discussions during symposium sessions.