Stefanie D. Krämer

Absorption prediction from physicochemical parameters.

Modern drug design not only focuses on the pharmacological activity of a compound but also considers its ability to be absorbed and to reach its site of action. The prediction of in vivo barrier permeabilities, particularly intestinal absorption and blood-brain barrier passage, are substantial concerns in the development of new drug compounds. For several decades, n-octanol-water partition coefficients dominated absorption prediction. In recent years, the basic physicochemical parameters describing both membrane permeability and lipophilicity have been established. This review provides an outline of some selected absorption-prediction models with emphasis on intestinal absorption.

No entry for TAT(44-57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides.

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism. We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb(3+)-containing liposomes. Upon chelation of Tb(3+) by an aromatic carboxylic acid, the fluorescence of Tb(3+) increases many fold. The CPP TAT(44-57)-SAM and TAT(37-53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol. In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44-57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44-57). Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44-57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively.

Lipid membrane interactions of indacaterol and salmeterol: Do they influence their pharmacological properties?

This study compares the lipid membrane interactions of indacaterol, an ultra long acting beta-2 agonist that is given once a day, to salmeterol, a twice a day beta-2 agonist, in order to elucidate the potential mechanisms leading to their different pharmacological properties. Salmeterol but not indacaterol perturbed dimyristoyl-phosphatidylcholine membranes. While the liposome partitioning of the two compounds was similar, independent of the lipid composition, the membrane affinity of indacaterol was two-fold greater than that of salmeterol when rafts, i.e. detergent-insoluble membrane domains, were used as the partition phase. The observed association kinetics with immobilized liposomes at physiological pH were two times faster for indacaterol than for salmeterol. A new model to explain the relationships between the drug/membrane interactions and drugs pharmacological properties considering multiple factors is proposed. The synergy between the higher partitioning of indacaterol into the raft micro domains and the faster membrane permeation of indacaterol could explain the faster onset and longer duration of therapeutic effect of indacaterol. The higher fluidizing effect of salmeterol on membrane fluidity may contribute to its lower intrinsic efficacy compared to indacaterol.

Towards the Predictability of Drug-Lipid Membrane Interactions: The pH-Dependent Affinity of Propranolol to Phosphatidylinositol Containing Liposomes

AbstractPurpose. Prediction of the pH-dependent affinity of (RS)-[3H]propranolol to mixed phosphatidylcholine (PhC)/phosphatidylinositol(Phl) membranes from the partitioning in the single lipid liposome/buffer systems. Methods. Partition studies in liposome/buffer systems were performed by means of equilibrium dialysis at 37°C between pH 2 and 11 at a molar propranolol to lipid ratio of 10−6 to 10−5 in the membrane. Results. The Phl membrane more strongly attracts the protonated (RS)-[3H]propranolol than the neutral solute, i.e. the partition coefficient of the protonated base (Pi) is 17′430 ± 1320, P of the neutral compound (Pn) is 3110 ± 1650. In the PhC-liposome system Pi is 580 ± 17, Pn 1860 ± 20. The partition coefficients show an exponential dependence on the molar Phl fraction in mixed liposomes. The partitioning in mixed PhC/Phl membranes is predictable from Pn and Pi in the single lipid liposome systems. Conclusions. The negative charge of biological lipid membranes causes strong electrostatic interactions with positively charged solutes. This strong attraction is not predictable from the octanol/buffer partition system, but it is important regarding drug accumulation in the tissue and drug attraction by certain lipids in the vicinity of membrane proteins.

Particle size and activation threshold: a new dimension of danger signaling

Previous studies have shown that single-stranded RNA (ssRNA) mixed with protamine forms particles and activates immune cells through Toll-like receptors (TLRs). We have found that the size of protamine-RNA particles generated depends on the electrolyte content when mixing the 2 components. Moreover, we have evidenced that (1) nanometric particles induce production of interferon-alpha, whereas (2) micrometric particles mainly induce production of tumor necrosis factor-alpha (TNF-alpha) in human immune cells. We found that the mechanisms underlying these observations are (1) nanoparticles but not microparticles are selectively phagocytosed by plasmacytoid dendritic cells (pDCs), which produce interferon-alpha and (2) monocytes that produce TNF-alpha have a higher activation threshold than that of pDCs. Thus, at the same time as sensing pathogen-associated molecular patterns such as ssRNA, the immune system distinguishes the size of the associated structure in such a way as to trigger the adapted antivirus (nanometric) or antibacterial/antifungal (micrometric) immune response. Our results introduce a new dimension in danger signaling--how size qualitatively affects innate response.

Evidence-based approach to assess passive diffusion and carrier-mediated drug transport.

Evidence supporting the action of passive diffusion and carrier-mediated (CM) transport in drug bioavailability and disposition is discussed to refute the recently proposed theory that drug transport is CM-only and that new transporters will be discovered that possess transport characteristics ascribed to passive diffusion. Misconceptions and faulty speculations are addressed to provide reliable guidance on choosing appropriate tools for drug design and optimization.

The biochemistry of drug metabolism--an introduction: part 4. reactions of conjugation and their enzymes.

This review continues a general presentation of the metabolism of drugs and other xenobiotics begun in three recent issues of Chemistry & Biodiversity. The present Part is dedicated to reactions of conjugation, namely methylation, sulfonation, and phosphorylation, glucuronidation and other glycosidations, acetylation and other acylations, the formation and fate of coenzyme A conjugates, glutathione conjugation, and the reaction of amines with carbonyl compounds. It presents the many transferases involved, their nomenclature, relevant biochemical properties, catalytic mechanisms, and the reactions they catalyze. Nonenzymatic reactions, mainly of glutathione conjugation, also receive due attention. A number of medicinally, environmentally, and toxicologically relevant examples are presented and discussed.

MDCK cell cultures as an epithelial in vitro model : Cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy

AbstractPurpose. Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. Methods. Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, α-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. Results. TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0−1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2−6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7−14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV (≥ 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. Conclusions. Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitromodel to study drug transport and/or interactions with drugs and excipients.

[18F]fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting.

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-β isoform) which are involved in inflammatory and autoimmune diseases, but its expression in healthy tissues and organs is highly restricted to only a few sites (e.g kidneys). Therefore, the FR is a promising target for imaging and therapy of cancer and inflammation using folate-based radiopharmaceuticals. Herein, we report the synthesis and evaluation of a novel folic acid conjugate with improved properties suitable for positron emission tomography (PET). [(18)F]-fluoro-deoxy-glucose folate ([(18)F]3) was synthesized based on the click chemistry approach using 2-deoxy-2-[(18)F]fluoroglucopyranosyl azide and a folate alkyne derivative. The novel radiotracer [(18)F]3 was produced in good radiochemical yields (25% d.c.) and high specific radioactivity (90 GBq/μmol). Compared to previously published (18)F-folic acid derivatives, an increase in hydrophilicity was achieved by using a glucose entity as a prosthetic group. Biodistribution and PET imaging studies in KB tumor-bearing mice showed a high and specific uptake of the radiotracer in FR-positive tumors (10.03 ± 1.12%ID/g, 60 min p.i.) and kidneys (42.94 ± 2.04%ID/g, 60 min p.i.). FR-unspecific accumulation of radioactivity was only found in the liver (9.49 ± 1.13%ID/g, 60 min p.i.) and gallbladder (17.59 ± 7.22%ID/g, 60 min p.i.). No radiometabolites were detected in blood, urine, and liver tissue up to 30 min after injection of [(18)F]3. [(18)F]-fluoro-deoxy-glucose-folate ([(18)F]3) is thus a promising PET radioligand for imaging FR-positive tumors.

Pharmacokinetics and safety of panobacumab : specific adjunctive immunotherapy in critical patients with nosocomial Pseudomonas aeruginosa O11 pneumonia.

OBJECTIVES Nosocomial Pseudomonas aeruginosa pneumonia remains a major concern in critically ill patients. We explored the potential impact of microorganism-targeted adjunctive immunotherapy in such patients. PATIENTS AND METHODS This multicentre, open pilot Phase 2a clinical trial (NCT00851435) prospectively evaluated the safety, pharmacokinetics and potential efficacy of three doses of 1.2 mg/kg panobacumab, a fully human monoclonal anti-lipopolysaccharide IgM, given every 72 h in 18 patients developing nosocomial P. aeruginosa (serotype O11) pneumonia. RESULTS Seventeen out of 18 patients were included in the pharmacokinetic analysis. In 13 patients receiving three doses, the maximal concentration after the third infusion was 33.9 ± 8.0 μg/mL, total area under the serum concentration-time curve was 5397 ± 1993 μg h/mL and elimination half-life was 102.3 ± 47.8 h. Panobacumab was well tolerated, induced no immunogenicity and was detected in respiratory samples. In contrast to Acute Physiology and Chronic Health Evaluation II (APACHE II) prediction, all 13 patients receiving three doses survived, with a mean clinical resolution in 9.0 ± 2.7 days. Two patients suffered a recurrence at days 17 and 20. CONCLUSIONS These data suggest that panobacumab is safe, with a pharmacokinetic profile similar to that in healthy volunteers. It was associated with high clinical cure and survival rates in patients developing nosocomial P. aeruginosa O11 pneumonia. We concluded that these promising results warrant further trials.