European Journal of Pharmaceutics and Biopharmaceutics (v.60, #2)

APV Calendar (S1-S2).

The commercially available reconstructed human epidermis models EpiSkin®, SkinEthic® and EpiDerm® demonstrate reasonable similarities to the native human tissue in terms of morphology, lipid composition and biochemical markers. These models have been identified as useful tools for the testing of phototoxicity, corrosivity and irritancy, and test protocols have been developed for such applications. For acceptance of these tests by the authorities, prevalidation or validation studies are currently in progress. Furthermore, first results also indicate their suitability for transport experiments of drugs and other xenobiotics across skin. Still, however, the barrier function of these reconstructed human epidermis models appears to be much less developed compared to native skin. Further adaptation of the models to the human epidermis, especially concerning the barrier function, therefore remains an important challenge in this area of research.
Keywords: Reconstructed human epidermis; Lipid composition; Biochemical markers; Drug delivery; Skin;

Transdermal iontophoresis: combination strategies to improve transdermal iontophoretic drug delivery by Yiping Wang; Rashmi Thakur; Qiuxi Fan; Bozena Michniak (179-191).
For several decades, there has been interest in using the skin as a port of entry into the body for the systemic delivery of therapeutic agents. However, the upper layer of the skin, the stratum corneum, poses a barrier to the entry of many therapeutic entities. Given a compound, passive delivery rate is often dependent on two major physicochemical properties: the partition coefficient and solubility. The use of chemical enhancers and modifications of the thermodynamic activity of the applied drug are two frequently employed strategies to improve transdermal permeation. Chemical enhancers are known to enhance drug permeation by several mechanisms which include disrupting the organized intercellular lipid structure of the stratum corneum , ‘fluidizing’ the stratum corneum lipids , altering cellular proteins, and in some cases, extracting intercellular lipids . However, the resulting increase in drug permeation using these techniques is rather modest especially for hydrophilic drugs. A number of other physical approaches such as iontophoresis, sonophoresis, ultrasound and the use of microneedles are now being studied to improve permeation of hydrophilic as well as lipophilic drugs. This article presents an overview of the use of iontophoresis alone and in conjunction with other approaches such as chemical enhancement, electroporation, sonophoresis, and use of microneedles and ion-exchange materials.
Keywords: Transdermal; Iontophoresis; Electroporation; Chemical enhancers; Sonophoresis; Microneedles; Ion-exchange materials;

Recent developments in delivering drugs to the lung are driving the need for in vitro methods to evaluate the fate of inhaled medicines. Constraints on experimentation using animals have promoted the use of human respiratory epithelial cell cultures to model the absorption barrier of the lung; with two airway cell lines, 16HBE14o- and Calu-3, and primary cultured human alveolar type I-like cells (hAEpC) gaining prominence. These in vitro models develop permeability properties which are comparable to those reported for native lung epithelia. This is in contrast to the high permeability of the A549 human alveolar cell line, which is unsuitable for use in drug permeability experiments. Tabulation of apparent permeability coefficients (P app) of compounds measured in ‘absorptive’ and ‘secretory’ directions reveals that fewer compounds (<15) have been evaluated in 16HBE14o- cells and hAEpC compared to Calu-3 cells (>50). Vectorial (asymmetric) transport of compounds is reported in the three cell types with P-glycoprotein, the most studied transport mechanism, being reported in all. Progress is being made towards in vitro–in vivo-correlation for pulmonary absorption and in the use of cultured respiratory cells to evaluate drug metabolism, toxicity and targeting strategies. In summary, methods for the culture of human respiratory epithelial cell layers have been established and data regarding their permeability characteristics and suitability to model the lung is becoming available. Discerning the circumstances under which the use of human respiratory cell models will be essential, or offers advantages over non-organ, non-species specific cell models, is the next challenge.
Keywords: Bronchial epithelial cells; Alveolar epithelial cells; Pulmonary drug delivery; In vitro models;

Cell culture models of the ocular barriers by Margit Hornof; Elisa Toropainen; Arto Urtti (207-225).
The presence of tight barriers, which regulate the environment of ocular tissues in the anterior and posterior part of the eye, is essential for normal visual function. The development of strategies to overcome these barriers for the targeted ocular delivery of drugs, e.g. to the retina, remains a major challenge. During the last years numerous cell culture models of the ocular barriers (cornea, conjunctiva, blood–retinal barrier) have been established. They are considered to be promising tools for studying the drug transport into ocular tissues, and for numerous other purposes, such as the investigation of pathological ocular conditions, and the toxicological screening of compounds as alternative to in vivo toxicity tests. The further development of these in vitro models will require more detailed investigations of the barrier properties of both the cell culture models and the in vivo ocular barriers. It is the aim of this review to describe the current status in the development of cell culture models of the ocular barriers, and to discuss the applicability of these models in pharmaceutical research.
Keywords: Cell culture model; Blood–retinal barrier; Blood–aqueous barrier; Cornea; Conjunctiva; Retinal pigment epithelium; Retinal capillary endothelium; Ocular drug delivery; Ocular absorption;

Conjunctiva plays many roles including protection of ocular surface, production of tear film, and a conduit for drug clearance (depending on drug properties) into the systemic circulation or for drug transport to the deep tissues of the eye. The conjunctiva, which is a moderately tight epithelium, endowed with various transport processes for the homeostasis of ions, solutes, and water in the conjunctival surface and tear film. Modulation of ion transport in the conjunctiva leads to alterations in transconjunctival fluid flow that may become useful for treatment of dry-eye state in the eye. As a possible drug delivery route to the posterior portion of the eye, conjunctiva is an attractive route due to both larger surface area than that of cornea and expression of several key transport processes. Tear contains d-glucose and many amino acids, in addition to the usual ions in the body fluids. Several ion-coupled solute transport processes for absorption of amino acids, d-glucose, monocarboxylate, nucleosides, and dipeptides are expressed in the conjunctiva. Thanks to the rich endowment of these transport processes, drug transport across the conjunctiva into the intraocular tissues may become quite feasible. Subconjunctival injection of microparticles and matrix materials (which allows sustained release of drugs) is shown to maintain reasonable levels of various drugs in the vitreous, perhaps attesting to the fact that conjunctiva per se may contribute as a part of multiple transport barrier(s) in ocular drug delivery. In addition, several conjunctival approaches have been investigated to optimize treatment of dry-eye syndrome and intraocular diseases, and more can be accomplished in the coming years.
Keywords: Conjunctival drug delivery; Carrier-mediated transport; Ion transport; Solute transport; Endocytosis; Subconjunctival administration; Iontophoresis;

Delivery aspects of small peptides and substrates for peptide transporters by Bente Steffansen; Carsten Uhd Nielsen; Sven Frokjaer (241-245).
The present summary highlight chemical strategies applied to improve plasma half-lives and oral bioavailability of peptidic drugs as well as view on intestinal and pancreatic peptidase mediated degradation of peptidic drugs. In general chemical strategies used to increase the oral bioavailability of peptidic drugs consisting of more than three amino acids is disappointing. On the other hand chemical approaches to stabilize peptidic drugs against metabolism seem promising for increasing plasma half-lives of parental peptidic drugs as well as for increasing oral bioavailability of di/tripeptidomimetics and dipeptidyl pro-moieties targeting peptide transporters.
Keywords: Proteolytic enzymes; Exopeptidases; Intestinal peptide transporters; Amide bond replacement; Peptides; Drug delivery; Prodrugs;

Polyethylenimine-based non-viral gene delivery systems by U. Lungwitz; M. Breunig; T. Blunk; A. Göpferich (247-266).
Gene therapy has become a promising strategy for the treatment of many inheritable or acquired diseases that are currently considered incurable. Non-viral vectors have attracted great interest, as they are simple to prepare, rather stable, easy to modify and relatively safe, compared to viral vectors. Unfortunately, they also suffer from a lower transfection efficiency, requiring additional effort for their optimization. The cationic polymer polyethylenimine (PEI) has been widely used for non-viral transfection in vitro and in vivo and has an advantage over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. Here, we give some insight into strategies developed for PEI-based non-viral vectors to overcome intracellular obstacles, including the improvement of methods for polyplex preparation and the incorporation of endosomolytic agents or nuclear localization signals. In recent years, PEI-based non-viral vectors have been locally or systemically delivered, mostly to target gene delivery to tumor tissue, the lung or liver. This requires strategies to efficiently shield transfection polyplexes against non-specific interaction with blood components, extracellular matrix and untargeted cells and the attachment of targeting moieties, which allow for the directed gene delivery to the desired cell or tissue. In this context, materials, facilitating the design of novel PEI-based non-viral vectors are described.
Keywords: Polyethylenimine; PEI; Intracellular trafficking; Endosomolytic peptides; Nuclear targeting; Cell targeting;

Nature's design of nanomotors by Pia D. Vogel (267-277).
The need for movement is an essential concept of all living organisms. On a macroscopic scale, animals and microbes have to be able to move towards food and away from poison and predators. Plants turn their leaves toward their energy source, the sunlight. But even on a molecular scale, movement is essential for life. It has been known for a long time that enzymes and proteins undergo large conformational changes while performing their biological tasks. The catalytically active regions of enzymes need to sequentially open to bind their respective substrates and close to allow the specific chemical reaction to occur in a defined chemical environment. The active sites finally open up again up to allow the product to be released. Molecular motors are proteins and protein complexes that have evolved in living cells to carry out a variety of functions essential for survival, reproduction and differentiation of the cells and organisms. They use chemical, electrochemical or potential energy and transduce that energy into physical, chemical or mechanical force. In this paper we review some of the molecular motors that were designed by nature to either perform physical work or that contain motor-like movements as part of their catalytic mechanism.
Keywords: Molecular motors; Rotary motors; Linear motors; ATPase; Dynein; Kinesin; Myosin;

Cryoconserved shielded and EGF receptor targeted DNA polyplexes: cellular mechanisms by Katharina von Gersdorff; Manfred Ogris; Ernst Wagner (279-285).
Recently, cryoconservable polyethylene glycol (PEG)-shielded and epidermal growth factor receptor (EGFR)-targeted polyplexes (EGF+ polyplexes) were engineered in our laboratory for tumor-directed transfer and expression of DNA. Here, we further analyzed specificity and kinetics of EGFR-mediated cellular uptake of these polyplexes. Similar to our previous results, EGF+ polyplexes significantly enhanced the transfection efficiency as compared to polyplexes without EGF (EGF polyplexes) in HUH-7 hepatoma cells and Renca-EGFR renal carcinoma cells. EGF+ polyplexes rapidly associated with the cells within 30 min of exposure, and binding of EGF+ polyplexes to the cells after 4 h was significantly higher than that of EGF polyplexes. In the presence of free EGF, both cell association and transfection efficiency of EGF+ polyplexes were markedly reduced indicating that these effects were primarily mediated via ligand receptor interaction. Fluorescence microscopy revealed that the cell-associated EGF+ polyplexes aggregated to micrometer sized clusters, resembling typical clustering of receptors upon ligand binding. In conclusion, EGFR-targeting enhances transfection efficiency due to accelerated and increased cell association followed by aggregation of the bound EGF+ polyplexes.
Keywords: DNA complexes; Non-viral gene transfer; Polyethylenimine; Polyethylene glycol; Transfection;

Cationic lipid–protamine–DNA (LPD) complexes for delivery of antisense c-myc oligonucleotides by Monika Junghans; Stefan M. Loitsch; Sebastian C.J. Steiniger; Jörg Kreuter; Andreas Zimmer (287-294).
In the present study, cationic lipid–peptide–DNA-complexes (LPDs) consisting of AH-Chol-liposomes and protamine–phosphodiester–oligonucleotide-particles (proticles) were introduced as carriers for antisense therapy. The LPDs were physically characterized, and a possible mechanism for adsorption of oligonucleotides (ODNs) was suggested. An increase in stability of ODNs against DNase I and serum nuclease digestion by these carriers was demonstrated. The hydrodynamic diameter increased after incubation with FCS which could be attributed to a protein coating of the particle surface. However, in cell culture medium lower particle sizes of the complexes occurred. In an antisense c-myc in vitro model, the effect of LPDs was tested using U937 cells. The C-MYC level was reduced after treatment of these antisense ODN carrier complexes. Furthermore, no changes in target mRNA concentration of the treated cells was found by reverse transcription and competitive multiplex-PCR.
Keywords: Protamine; Nanoparticles; Liposomes; Antisense oligonucleotides;

Assessing transferrin modification of liposomes by atomic force microscopy and transmission electron microscopy by Samah Anabousi; Michael Laue; Claus-Michael Lehr; Udo Bakowsky; Carsten Ehrhardt (295-303).
Site-specific delivery of drugs and therapeutics can significantly reduce drug toxicity and increase the therapeutic effect. Transferrin (Tf) is one suitable ligand to be conjugated to drug delivery systems to achieve site-specific targeting, due to its specific binding to transferrin receptors (TfR), expressed on several cell types of therapeutic interest. TfRs have been reported to be highly expressed on the surfaces of tumour cells and the well-characterised and efficient mechanism of internalisation of Tf has been exploited for the delivery of anticancer drugs, proteins, and therapeutic genes into primarily proliferating malignant cells. Liposomes are effective vehicles for drugs, genes and vaccines and can be easily modified with proteins, antibodies, and other appropriate ligands, resulting in attractive formulations for targeted drug delivery. In this study, we used atomic force microscopy (AFM) and transmission electron microscopy (TEM) to confirm the conjugation of Tf to liposomes by three different coupling methods. In addition, the conventional assays for quantification of protein amount (BCA) and phospholipid content (according to Steward) were performed. AFM and TEM were able to display Tf-molecules on the liposomal surfaces and can be routinely used to obtain additional visual information on the protein–drug carrier conjugation in a fast and reliable manner.
Keywords: Drug targeting; Transferrin; Atomic force microscopy; Transmission electron microscopy; Anticancer therapy; Liposomes;

Human cornea construct HCC—an alternative for in vitro permeation studies? A comparison with human donor corneas by Stephan Reichl; Stefanie Döhring; Jürgen Bednarz; Christel C. Müller-Goymann (305-308).
Transcorneal in vitro permeation studies of ophthalmic drugs are normally performed with either excised animal corneas or latterly corneal cell culture models. A good correlation between these models and excised animal corneas regarding permeation behaviour of drugs has already been shown. However, comparisons between corneal in vitro models containing human cells and excised human corneas do not exist yet. Therefore in the present study the transcorneal permeation of six different model drugs (pilocarpine hydrochloride, befunolol hydrochloride, hydrocortisone, diclofenac sodium, clindamycin hydrochloride and timolol maleate) across our previously described three-dimensional organotypic human cornea construct (HCC) was tested using Franz diffusion cells and compared with permeation data obtained from human donor corneas. The HCC showed a similar permeation behaviour compared with human donor cornea for all substances. The permeabilities (permeation coefficients P) of the human cornea equivalent versus the human donor cornea were the same in the case of diclofenac, clindamycin, timolol, but marginally decreased for hydrocortisone and slightly increased for pilocarpine and befunolol. These small differences of permeation coefficients were expressed as factors and only varied from 0.8 to 1.4. The results indicate that the HCC may be an alternative for in vitro permeation studies and appropriate for predicting drug absorption into the human eye.
Keywords: Human cornea equivalent; Cell culture; In vitro model; Drug permeation studies; Human donor cornea;

In vitro–in vivo correlation (IV–IVC) is the relationship between an in vitro parameter (drug release or other rheological properties/measurement such as viscosity and spreadability) and an in vivo parameter (pharmacodynamic (PD) or dermatopharmacokinetic (DPK) or other measurement). In a true sense of correlation, in vitro measurement should predict in vivo performance of the product. For topically applied preparations, one of the in vitro measurements is the drug release from the formulation and in vivo measurement is the drug concentration in the stratum corneum, DPK or the PD measurements. The in vitro release of the drug is the property of the dosage form and is a measure of product quality and ‘sameness’, especially after certain Scale-UP and Post Approval Changes after initial drug approval.To obtain an IV–IVC for a topically applied drug product is a difficult challenge. However, some success has been achieved in showing a relationship between the drug release and PD and/or DPK measurement. Interestingly, one of the in vitro rheological properties was found to relate to the observed PD and DPK response for Clobetasol dipropionate products. Different rheological properties of the two formulation products explained the difference in DPK results obtained by two laboratories for the same tretinoin gel products. In the scientific arena, it is difficult to obtain a classical IV–IVC even for orally administered products and is more so difficult for topically administered drug products.
Keywords: In vitro–in vivo correlation; In vitro release; Rheological properties; Spreadability; Dermatopharmacokinetics; DPK; Pharmacodynamics;

Doctoral Theses (317-318).