Improved cryopreservation media formulation reduces costs of maintenance while preserving function of genetically modified insect cells.

Insect cell lines are an invaluable resource that facilitate various fundamental and applied research programs. Genetically engineered insect cell lines, in particular, serve as a platform through which the function of heterologously expressed proteins can be studied. However, a barrier to more widespread utilization and distribution of insect cell lines, genetically modified or not, is the technical and operational challenge associated with traditional cryopreservation methods, including their dependence on the use of liquid nitrogen facilities, animal or human serum products, and relatively high concentrations of permeating cryoprotectants (e.g., DMSO). Recent innovations in cryopreservation technologies have produced reagents with improved abilities to effectively preserve mammalian cell lines for long periods in regular laboratory deep freezers without using serum products, but their effectiveness in preserving genetically engineered insect cell lines has not yet been evaluated. In this study, we engineered Sf9 cells to express a dopamine receptor and used them as a model for evaluating the efficacy of a novel cryopreservation medium product, C80EZ®-INSECT, in not only preserving cell viability and proliferation efficiency but also maintaining the insect cell line's "functionality" after storage at -80°C. We found that the engineered Sf9 cells frozen using C80EZ®-INSECT with 5% DMSO alone and stored at -80°C for 6 mo displayed higher viability and growth rates than cells frozen using traditional fetal bovine serum (FBS)-based cryopreservation media with 10% DMSO that were stored at -80°C or in liquid nitrogen for the same period of time. We also found that after 6 mo of storage at -80°C or in liquid nitrogen the cells retained a responsiveness to dopamine comparable to that of the initial cell line, regardless of the cryopreservation reagent used. These results suggest that, due to the unique characteristics of C80EZ®-INSECT in preventing ice recrystallization and reducing ice crystal size and cellular apoptosis during cryostorage procedures, it is an effective cryopreservation reagent for genetically engineered Sf9 cells, and it practically eliminates the need for liquid nitrogen-based storage facilities and FBS-based cryopreservation formulations, as well as reduces the use of permeating cryoprotectants.

Design, synthesis and biological assessment of new selective COX-2 inhibitors including methyl sulfonyl moiety.

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause peptic lesions in the gastrointestinal mucosa by inhibiting the cyclooxygenase-1 (COX-1) enzyme. Selective COX-2 inhibition causes decreased side effects over current NSAIDs. Therefore, the studies about selective inhibition of COX-2 enzyme are very important for new drug development. The design, synthesis and biological activity evaluation of novel derivatives bearing thiazolylhydrazine-methyl sulfonyl moiety as selective COX-2 inhibitors were aimed in this paper. The structures of synthesized compounds were assigned using different spectroscopic techniques such as 1H NMR, 13C NMR and HRMS. In addition, the estimation of ADME parameters for all compounds was carried out using in silico process. The evaluation of in vitro COX-1/COX-2 enzyme inhibition was applied according to the fluorometric method. According to the enzyme inhibition results, synthesized compounds showed the selectivity against COX-2 enzyme inhibition as expected. Compounds 3a, 3e, 3f, 3g, 3i and 3j demonstrated significant COX-2 inhibition potencies. Among them, compound 3a was found to be the most effective derivative with an IC50 value of 0.140 ± 0.006 µM. Moreover, it was seen that compound 3a displayed a more potent inhibition profile at least 12-fold than nimesulide (IC50 = 1.684 ± 0.079 µM), while it showed inhibitory activity at a similar rate of celecoxib (IC50 = 0.132 ± 0.005 µM). Molecular modelling studies aided in the understanding of the interaction modes between this compound and COX-2 enzyme. It was found that compound 3a had a significant binding property. In addition, the selectivity of obtained derivatives on COX-2 enzyme could be explained and discussed by molecular docking studies.

Cellulose Dissolution in Mixtures of Ionic Liquids and Dimethyl Sulfoxide: A Quantitative Assessment of the Relative Importance of Temperature and Composition of the Binary Solvent.

We studied the dissolution of microcrystalline cellulose (MCC) in binary mixtures of dimethyl sulfoxide (DMSO) and the ionic liquids: allylbenzyldimethylammonium acetate; 1-(2-methoxyethyl)-3-methylimidazolium acetate; 1,8-diazabicyclo [5.4.0]undec-7-ene-8-ium acetate; tetramethylguanidinium acetate. Using chemometrics, we determined the dependence of the mass fraction (in %) of dissolved cellulose (MCC-m%) on the temperature, T = 40, 60, and 80 °C, and the mole fraction of DMSO, χDMSO = 0.4, 0.6, and 0.8. We derived equations that quantified the dependence of MCC-m% on T and χDMSO. Cellulose dissolution increased as a function of increasing both variables; the contribution of χDMSO was larger than that of T in some cases. Solvent empirical polarity was qualitatively employed to rationalize the cellulose dissolution efficiency of the solvent. Using the solvatochromic probe 2,6-dichloro-4-(2,4,6-triphenylpyridinium-1-yl)phenolate (WB), we calculated the empirical polarity ET(WB) of cellobiose (a model for MCC) in ionic liquid (IL)-DMSO mixtures. The ET(WB) correlated perfectly with T (fixed χDMSO) and with χDMSO (fixed T). These results show that there is ground for using medium empirical polarity to assess cellulose dissolution efficiency. We calculated values of MCC-m% under conditions other than those employed to generate the statistical model and determined the corresponding MCC-m% experimentally. The excellent agreement between both values shows the robustness of the statistical model and the usefulness of our approach to predict cellulose dissolution, thus saving time, labor, and material.

Lithium Hexamethyldisilazane Transformation of Transiently Protected 4-Aza/Benzimidazole Nitriles to Amidines and their Dimethyl Sulfoxide Mediated Imidazole Ring Formation.

Trimethylsilyl-transient protection successfully allowed the use of lithium hexamethyldisilazane to prepare benzimidazole (BI) and 4-azabenzimidazole (azaBI) amidines from nitriles in 58-88% yields. This strategy offers a much better choice to prepare BI/azaBI amidines than the lengthy, low-yielding Pinner reaction. Synthesis of aza/benzimidazole rings from aromatic diamines and aldehydes was affected in dimethyl sulfoxide in 10-15 min, while known procedures require long time and purification. These methods are important for the BI/azaBI-based drug industry and for developing specific DNA binders for expanded therapeutic applications.

Low-cost and broadband terahertz antireflection coatings based on DMSO-doped PEDOT/PSS.

We report the potential application of 6% dimethylsulfoxide (DMSO)-doped poly (3, 4-ethylenedioxythiophene)/poly (4-styrenesulfonate) (PEDOT/PSS) as a low cost and broadband terahertz (THz) antireflection coating based on the impedance matching effect. The reflected pulses from the quartz and silicon substrates are observed to change with the thickness of the PEDOT/PSS layer. Theoretical analysis based on an equivalent transmission line circuit model and FDTD computational simulations have been used to understand the experimental results. Excellent impedance matching is achieved by a ∼39-nm-thick 6% DMSO-doped PEDOT/PSS layer on quartz, and a ∼101-nm-thick 6% DMSO-doped PEDOT/PSS layer on silicon due to the almost-frequency-independent conductivity of the thin film between 0.3 and 2.5 THz. In the critical conditions, the normalized main pulse transmission remains as high as 74% and 64%, for the quartz and silicon substrates, respectively, significantly higher than the existing state of the art THz antireflection coatings.

Thermodynamics of mixing water with dimethyl sulfoxide, as seen from computer simulations.

The Helmholtz free energy, energy, and entropy of mixing of eight different models of dimethyl sulfoxide (DMSO) with four widely used water models are calculated at 298 K over the entire composition range by means of thermodynamic integration along a suitably chosen thermodynamic path, and compared with experimental data. All 32 model combinations considered are able to reproduce the experimental values rather well, within RT (free energy and energy) and R (entropy) at any composition, and quite often the deviation from the experimental data is even smaller, being in the order of the uncertainty of the calculated free energy or energy, and entropy values of 0.1 kJ/mol and 0.1 J/(mol K), respectively. On the other hand, none of the model combinations considered can accurately reproduce all three experimental functions simultaneously. Furthermore, the fact that the entropy of mixing changes sign with increasing DMSO mole fraction is only reproduced by a handful of model pairs. Model combinations that (i) give the best reproduction of the experimental free energy, while still reasonably well reproducing the experimental energy and entropy of mixing, and (ii) that give the best reproduction of the experimental energy and entropy, while still reasonably well reproducing the experimental free energy of mixing, are identified.

A refinement to the liquidus-tracking method for vitreous preservation of articular cartilage.

The recent liquidus-tracking method developed by Pegg et al. (2006a), as an alternative pathway to vitrification, achieved reasonable survival of post-thawing chondrocytes in situ. One of the main drawbacks of this method is the long duration of the cryoprotectant addition/removal process. This study was conducted to investigate the possibility of reducing the time by rationalizing the final dimethyl sulfoxide (Me2SO) concentration loaded in tissue before being plunged into liquid nitrogen. Using the differential scanning calorimetric technique, the critical cooling and warming rates for solutions of Me2SO in CPTes2 (a potassium-rich medium, modified slightly from Taylor's original formulation by Pegg et al.) were obtained. The critical cooling and warming rates for 47.5 percent (w/w) solution are < 2.5 degree C per min and < 10 degree C per min, respectively, which could be readily realized for 4 ml solution samples held in polypropylene cryovials as demonstrated by experiments. For articular cartilage, 47.5 percent (w/w) may be recommended as the final concentration of Me2SO loaded in the tissue, which will lead to a time cut of about one-third compared with the original protocol of Pegg et al. (2006a).

Assessment of the integrity of compounds stored in assay-ready plates using a kinase sentinel assay.

Sentinel assays are a convenient adjunct to LC-MS purity assessment to monitor the integrity of compounds in pharmaceutical screening collections over time. To assess the stability of compounds stored both at room temperature and at -20°C in assay-ready plates that were either vacuum pack-sealed using a convenient industrial vacuum sealing method or individually sealed using conventional foil seals, a diverse collection of ~ 5,000 compounds was assayed using a robust biochemical kinase assay at intervals over a one year period. Assay results at each time point were compared to those of initial assay using a series of correlations of compound Percent of Control (POC) values as well as IC50 values of a subset of compounds in 200 nL or 500 nL plates. The fraction of hits in common between initial assays and assays at later time points ranged from 82% to 95% over one year and remained relatively constant over time with all storage temperatures or sealing methods tested. A majority of the hits that exhibited a consistent gradual trend to lower potency over one year storage were shifted to lower potency upon the rapid removal of DMSO solvent. Compound precipitation rather than compound decomposition is the likely reason for trends to lower potency for most such compounds over the storage period. Plates stored at room temperature featured a significantly higher fraction of hits that exhibited a trend to lower apparent potency than those stored at -20°C suggesting that this lower temperature is preferable for longer-term storage.

High-throughput quality control of DMSO acoustic dispensing using photometric dye methods.

One high-throughput technology gaining widespread adoption in industry and academia is acoustic liquid dispensing, in which focused sound waves eject nanoliter-sized droplets from a solution into a recipient microplate. This technology allows for direct dispensing of small-molecule compounds or reagents dissolved in DMSO, while keeping a low final concentration of organic solvent in an assay. However, acoustic dispensing presents unique quality control (QC) challenges when measuring the accuracy and precision of small dispense volumes ranging from 2.5 to 100 nL. As part of an effort to develop a rapid and cost-effective QC method for acoustic dispensing of 100% DMSO, we implemented the first high-throughput photometric dual-dye-based QC protocol in the nanoliter volume range. This technical note validates the new photometric 100% DMSO QC method and highlights its cost-effectiveness when compared with conventional low-throughput fluorimetric QC methods. In addition, a potential software solution is described for the analysis, storage, and display of accumulated high-throughput QC data, called LabGauge. As the need for high-throughput QC grows, conventional low-throughput methods can no longer meet demand. Validated high-throughput techniques, such as the dual-dye photometric method, will need to be implemented.

A hybrid neutron diffraction and computer simulation study on the solvation of N-methylformamide in dimethylsulfoxide.

The solvation of N-methylformamide (NMF) by dimethylsulfoxide (DMSO) in a 20% NMF/DMSO liquid mixture is investigated using a combination of neutron diffraction augmented with isotopic substitution and Monte Carlo simulations. The aim is to investigate the solute-solvent interactions and the structure of the solution. The results point to the formation of a hydrogen bond (H-bond) between the H bonded to the N of the amine group of NMF and the O of DMSO particularly strong when compared with other H-bonded liquids. Moreover, a second cooperative H-bond is identified with the S atom of DMSO. As a consequence of these H-bonds, molecules of NMF and DMSO are rather rigidly connected, establishing very stable dimmers in the mixture and very well organized first and second solvation shells.

Structure prediction, disorder and dynamics in a DMSO solvate of carbamazepine.

We have applied crystal structure prediction methods to understand and predict the formation of a DMSO solvate of the anti-convulsant drug carbamazepine (CBZ), in which the DMSO molecules are disordered. Crystal structure prediction calculations on the 1:1 CBZ:DMSO solvate revealed the generation of two similar low energy structures which differ only in the orientation of the DMSO molecules. Analysis of crystal energy landscapes generated at 0 K suggests the possibility of solvent disorder. A combined computational and experimental study of the changes in the orientation of the DMSO within the crystal structure revealed that the nature of the disorder changes with temperature. At low temperature, the DMSO disorder is static whilst at high temperature the DMSO configurations can interconvert by a 180° rotation of the DMSO molecules within the lattice. This 180° rotation of the DMSO molecules drives a phase change from a high temperature dynamically disordered phase to a low temperature phase with static disorder. Crystallisation of a DMSO solvate of the related molecule epoxycarbamazepine resulted in a different degree of DMSO disorder in the crystal structure, despite the similarity of the carbamazepine and epoxycarbamazepine molecules. We believe consideration of disorder and its contribution to entropy and crystal free energies at temperature other than 0 K is fundamental for the accuracy of future energy rankings in crystal structure prediction calculations of similar solvated structures.

Exploring solvent effects upon the Menshutkin reaction using a polarizable force field.

The energetics of the Menshutkin reaction between triethylamine and ethyl iodide have been computed using B3LYP and MP2 with the LANL2DZ, LANL2DZd, SVP, MIDI!, 6-311G(d,p), and aug-cc-PVTZ basis sets. Small- and large-core energy-consistent relativistic pseudopotentials were employed. Solvent effect corrections were computed from QM/MM Monte Carlo simulations utilizing free-energy perturbation theory, PDDG/PM3, and both a nonpolarizable OPLS and polarizable OPLS-AAP force field. The B3LYP/MIDI! theory level provided the best DeltaG(++) values with a mean absolute error (MAE) of 4.9 kcal/mol from experiment in cyclohexane, CCl(4), THF, DMSO, acetonitrile, water, and methanol. However, the relative rates in cyclohexane, and to a certain extent CCl(4), were determined to be greatly underestimated when using the nonpolarizable OPLS force field. An overall reduction in the MAE to 3.1 kcal/mol using B3LYP/MIDI!/OPLS-AAP demonstrated the need for a fully polarizable force field when computing solvent effects for highly dipolar transition structures in low-dielectric media. The MAEs obtained with PDDG/PM3/OPLS and OPLS-AAP of 5.3 and 3.8 kcal/mol, respectively, provided comparable results to B3LYP at a fraction of the computational resources. The large rate accelerations observed in the reaction were correlated to an increased stabilization of the emerging charge separation at the transition state via favorable solute-solvent interactions.

Retinoic acid and dimethyl sulfoxide promote efficient delivery of transgenes to mouse skin by topically transdermal penetration.

Simple and efficient gene transfer to the skin would facilitate many local and systemic gene therapy applications. This study reports a novel approach that allows expression of plasmid DNA in epidermis and hair follicle cells with dimethyl sulfoxide (DMSO) after pre-treatment with depilation and retinoic acid (RA) for the purposes of gene therapy. This study investigated the transdermal efficacy of gene to mouse skin when utilizing DMSO after RA pre-treatment. Retinoic acid pre-treatment can increase the efficiency of transfection. This finding indicates that one can more effectively and much less expensively make use of genes therapy to treat diseases of the hair and skin.

The assessment of chromophores in bleached cellulosic pulps employing UV-Raman spectroscopy.

UV-Resonance Raman (UV-RR) coupled with UV-visible Diffuse Reflectance (UV-vis DR) spectroscopy was applied to a solid-state study of chromophores in Eucalyptus globulus kraft cellulosic pulps bleached by chlorine dioxide and hydrogen peroxide. The UV-RR spectra were acquired at 325nm laser beam excitation, which was shown to be appropriate for selective analysis of chromophore structures in polysaccharides. The proposed approach allowed the monitoring of chromophores in pulps and to track the extent of polysaccharide oxidation. However, precaution was suggested while performing a quantitative analysis of chromophores at the characteristic band of approximately 1600cm(-1) because of charge transfer complexes (CTCs) that exist in the pulp. These CTCs can affect the intensity of the aforementioned band by diminishing the conjugate state in the chromophore moieties. The amount of carbonyl and carboxyl groups in polysaccharides correlated with the intensity of the band at 1093cm(-1). The analysis of UV-RR spectra revealed xylan as an important source of chromophores in eucalypt kraft pulp.

A computational study on cannabinoid receptors and potent bioactive cannabinoid ligands: homology modeling, docking, de novo drug design and molecular dynamics analysis.

When X-ray structure of a ligand-bound receptor is not available, homology models of the protein of interest can be used to obtain the ligand-binding cavities. The steroelectronic properties of these cavities are directly related to the performed molecular model coordinates. Thus, the use of different template structures for homology may result in variation of ligand-binding modes. We have recently reported the MD simulations of a potent CB ligand at bovine rhodopsin-based CB1 and CB2 receptors (Durdagi et al., Bioorg Med Chem 16:7377-7387, 2008). In this present study, a homology modeling study based on the beta2-adrenergic receptor for both CB1 and CB2 receptors was performed, and the results were compared with rhodopsin-based models. In addition, the role of membrane bilayers to the adopted conformations of potent AMG3 CB ligand has been analyzed for receptor-free and membrane-associated receptor systems. The performed MD trajectory analysis results have shown that gauche conformations at the terminal segment of the alkyl side chain of AMG3 are not favored in solution. Different adopting dihedral angles defined between aromatic and dithiolane rings at the active sites of the CB1 and CB2 receptors, which are adapted lead to different alkyl side chain orientations and thus, may give clues to the medicinal chemists to synthesize more selective CB ligands. The binding sites of receptors derived by rhodopsin-based models have been regenerated using the beta2-adrenergic based template receptors. The re-obtained models confirmed the ligand-binding pockets that were derived based on rhodopsin.

Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations.

BACKGROUND AND OBJECTIVE: Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically also contain gelatin-binding gelatinases that may cleave FN, reduce its stability and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN. MATERIAL AND METHODS: Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 m urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration. RESULTS: Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose, whereas subsequent elution with 4 m urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN, which was more stable than FN eluted by urea only. Fibronectin degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly. CONCLUSION: The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared with intact FN.

Assessment of the extent of starch dissolution in dimethyl sulfoxide by 1H NMR spectroscopy.

Complete dissolution is needed for the separation, characterization, or homogeneous labeling of whole starch molecules. A method is presented to quantify the extent of starch dissolution in DMSO for the first time; it is validated on a commercial rice starch. It is used directly on starch dispersions containing possible undissolved or co-dissolved species. High-amylose maize starches, known to be digested slowly in vivo, only quantitatively dissolve in the presence of high concentrations of an H-bond disrupter, LiBr, although they form clear dispersions at low LiBr concentrations. Starch quantitatively dissolves from waxy rice flours; non-starch components partially co-dissolve but do not interfere with the dissolution quantification.

In vitro assessment of aortic stent-graft integrity following exposure to Onyx liquid embolic agent.

PURPOSE: Endovascular stent-grafts are increasingly being used for treatment of abdominal and thoracic aortic aneurysms. Postprocedural complications include development of endoleaks. Recently, an embolic agent known as Onyx has been employed to treat type II endoleaks. Onyx is a biocompatible copolymer dissolved in dimethyl sulfoxide (DMSO). Although DMSO is known to damage some angiographic catheters, little is known concerning whether this compound damages stent-graft material. The current study was undertaken to directly explore this issue. MATERIALS AND METHODS: Four stent-grafts were evaluated: the Excluder, Zenith, AneuRx, and Talent. Stent-grafts were incubated for 24 hours at 37 degrees C under each of the following conditions: DMSO alone, 50/50 mixture of DMSO/Onyx, mixture of 1 part 50/50 DMSO/Onyx and 9 parts whole blood, and untreated control. Stent-grafts were microdissected into 15-mm sections, after which they were evaluated with scanning electron microscopy. RESULTS: No appreciable differences between stent-grafts exposed to DMSO and untreated controls were seen. Although liquid embolic agent was seen coating stent-grafts exposed to a 50/50 mixture of DMSO and Onyx, no evidence of fiber breakdown was noted. Stent-grafts exposed to DMSO/Onyx/whole blood demonstrated a thin coating of clot and Onyx without visual evidence of fiber compromise. CONCLUSIONS: The current study provides compelling evidence that short-term exposure of endograft material to DMSO, DMSO/Onyx, or DMSO/Onyx/whole blood is not associated with acute structural compromise of four commonly used aortic endografts. Future in vivo studies will help to further establish the safety of this agent.

Low-dimensional copper(II) complexes triply bridged with azide/carboxylate/DMSO showing very strong ferromagnetic interaction and influence of dipolar fields at low temperatures: a quantum Monte Carlo magnetic study.

Two one-dimensional (1D) azido, carboxylato and DMSO triply bridged coordination polymers of formula [Cu(mu1,1-N3)(mu1,3-(C4H3S-CH2COO))(mu-DMSO)]n (1) and [Cu3(mu1,1-N3)4(mu1,3-(C4H3S-COO)2)(mu-DMSO)2]n (2) have been synthesized, structurally characterized, and their magnetic properties have been studied. Cu(II) atoms 1 in are triply bridged by an EO type azide, a syn-syn carboxylate and a mu-O from a DMSO molecule. Complex 2 presents a trinuclear repeating unit in which the Cu(II) atoms are triply bridged in a fashion identical to 1, while the trinuclear moieties are linked through two centrosymmetrically related EO azides. The variable-temperature magnetic susceptibilities of complexes 1 and 2 have been measured in the range 2-300 K under various external fields in the range 0.02-1.0 T. Both curves are almost superimposable with small differences in the low temperature range and reflect the same ferromagnetic behaviour in almost all the temperature range. Quantum Monte Carlo (QMC) studies were carried out in order to fit the susceptibility curves of the two compounds. The simulated parameters are J = 126(2) cm(-1), g = 2.15(1) for compound and J1 = 126(2) cm(-1), J2 = 80(3) cm(-1)g = 2.17(1) for compound 2, proving that large ferromagnetic interactions exist in both compounds. EPR studies showed the temperature dependence of g-factors. The important g shift in the temperature range 4-100 K is attributed to internal dipolar magnetostatic fields.

Suitability of commonly used excipients for electrophysiological in-vitro safety pharmacology assessment of effects on hERG potassium current and on rabbit Purkinje fiber action potential.

INTRODUCTION: Regulatory guidelines require investigation of the liability for delayed ventricular repolarization by new chemical entities within a broad concentration range in-vitro. However, investigation can be limited by poor drug aqueous solubility, and by solvent physicochemical attributes that disrupt cell membrane integrity. Although excipients or solubilizing agents may aid to achieve the necessary high concentrations, no comprehensive overview on the suitability of solvents for in-vitro electrophysiological safety studies exists. METHODS: Excipients were tested for potential interference with the hERG (human ether-a-go-go-related gene) K(+) current (whole-cell voltage-clamp, 23+/-2 degrees C), and the shape of rabbit Purkinje fiber action potentials (conventional glass microelectrode technique, 37+/-1 degrees C). RESULTS AND DISCUSSION: Water-soluble complexation builders/carriers had little effect on hERG K(+) current at up to 50 mg/ml (BSA, bovine serum albumin) and 11 mg/ml (HP-beta-CD, hydroxypropyl-beta-cyclodextrin; IC(20), concentration of 20% inhibition). Water-soluble organic (co)solvents inhibited hERG K(+) currents (IC(20), %/mM): 0.7/152, ethanol; 0.9/67, Transcutol; 1.2/154, DMSO (dimethylsulfoxide); 1.6/389, acetonitrile; 1.9/48, polyethylene glycol 400; 2.1/660, methanol. Part of their inhibitory effect is attributed to the osmolality of extracellular solutions, because hERG IC(20) and extrapolated osmolality at the hERG IC(20) strongly correlate. Water-soluble non-ionic solubilizers/surfactants are potent inhibitors of hERG K(+) current with IC(20) concentrations of 0.07% (Cremophor EL) or lower (Tween 20, Tween 80: approximately 0.001%). Part of this inhibitory effect is attributed to their interaction with lipid membranes, because hERG inhibition occurs close to critical micelle concentrations (Cremophor, approximately 0.009%; Tween 20, approximately 0.007%). Purkinje fiber action potentials are little affected by HP-beta-CD at up to 2 mg/ml, while DMSO tends to shorten the action potential duration at 1%. CONCLUSION: When conducting electrophysiological in-vitro assessments of drug effects, solubilizers/surfactants (Cremophor EL, Tween 20, Tween 80) should be avoided. Instead, water-soluble organic (co)solvents (methanol, acetonitrile, DMSO) or complexation builders/carriers (HP-beta-CD, BSA) appear to be more favorable.