Amber Light Control of Peptide Secondary Structure by a Perfluoroaromatic Azobenzene Photoswitch.

The incorporation of photoswitches into the molecular structure of peptides and proteins enables their dynamic photocontrol in complex biological systems. Here, a perfluorinated azobenzene derivative triggered by amber light was site-specifically conjugated to cysteines in a helical peptide by perfluoroarylation chemistry. In response to the photoisomerization (trans→cis) of the conjugated azobenzene with amber light, the secondary structure of the peptide was modulated from a disorganized into an amphiphilic helical structure.

Harnessing Bile for Drug Absorption through Rational Excipient Selection.

Bile solubilization and apparent solubility at resorption sites critically affect the bioavailability of orally administered and poorly water-soluble drugs. Therefore, identification of drug-bile interaction may critically determine the overall formulation success. For the case of the drug candidate naporafenib, drug in solution at phase separation onset significantly improved with polyethylene glycol-40 hydrogenated castor oil (RH40) and amino methacrylate copolymer (Eudragit E) but not with hydroxypropyl cellulose (HPC) in both phosphate-buffered saline (PBS) and PBS supplemented with bile components. Naporafenib interacted with bile as determined by 1H and 2D 1H-1H nuclear magnetic resonance spectroscopy and so did Eudragit E and RH40 but not HPC. Flux across artificial membranes was reduced in the presence of Eudragit E. RH40 reduced the naporafenib supersaturation duration. HPC on the other side stabilized naporafenib's supersaturation and did not substantially impact flux. These insights on bile interaction correlated with pharmacokinetics (PK) in beagle dogs. HPC preserved naporafenib bile solubilization in contrast to Eudragit E and RH40, resulting in favorable PK.

Bile Is a Selective Elevator for Mucosal Mechanics and Transport.

Mucus mechanically protects the intestinal epithelium and impacts the absorption of drugs, with a largely unknown role for bile. We explored the impacts of bile on mucosal biomechanics and drug transport within mucus. Bile diffused with square-root-of-time kinetics and interplayed with mucus, leading to transient stiffening captured in Brillouin images and a concentration-dependent change from subdiffusive to Brownian-like diffusion kinetics within the mucus demonstrated by differential dynamic microscopy. Bile-interacting drugs, Fluphenazine and Perphenazine, diffused faster through mucus in the presence of bile, while Metoprolol, a drug with no bile interaction, displayed consistent diffusion. Our findings were corroborated by rat studies, where co-dosing of a bile acid sequestrant substantially reduced the bioavailability of Perphenazine but not Metoprolol. We clustered over 50 drugs based on their interactions with bile and mucin. Drugs that interacted with bile also interacted with mucin but not vice versa. This study detailed the dynamics of mucus biomechanics under bile exposure and linked the ability of a drug to interact with bile to its abbility to interact with mucus.

Bioactive Electrospun Fibers: Fabrication Strategies and a Critical Review of Surface-Sensitive Characterization and Quantification.

Fabricating a porous scaffold with high surface area has been a major strategy in the tissue engineering field. Among the many fabrication methods, electrospinning has become one of the cornerstone techniques due to its enabling the fabrication of highly porous fibrous scaffolds that are of natural or synthetic origin. Apart from the basic requirements of mechanical stability and biocompatibility, scaffolds are further expected to embody functional cues that drive cellular functions such as adhesion, spreading, proliferation, migration, and differentiation. There are abundant distinct approaches to introducing bioactive molecules to have a control over cellular functions. However, the lack of a thorough understanding of cell behavior with respect to the availability and spatial distribution of the bioactive molecules in 3D fibrous scaffolds is yet to be addressed. The rational selection of proper sets of characterization techniques would essentially impact the interpretation of the cell-scaffold interactions. In this timely Review, we summarize the most popular methods to introduce functional compounds to electrospun fibers. Thereafter, the strength and limitations of the conventional characterization methods are highlighted. Finally, the potential and applicability of emerging characterization techniques such as high-resolution/correlative microscopy approaches are further discussed.

Cyano(fluoro)borate and cyano(hydrido)borate ionic liquids: low-viscosity ionic media for electrochemical applications.

The synthesis and detailed characterization of low-viscosity room-temperature ionic liquids (RTILs) and [BnPh3P]+ salts with the cyano(fluoro)borate anions [BF(CN)3]- (MFB), [BF2(CN)2]- (DFB), and [BF3(CN)]- as well as the new mixed-substituted anion [BFH(CN)2]- (FHB) is described. The RTILs with [EMIm]+ or [BMPL]+ as countercations were obtained in yields of up to 98% from readily available alkali metal salts and in high purities that allow application in electrochemical devices. Trends in thermal stability, melting and freezing behavior, density, electrochemical stability, dynamic viscosity, specific conductivity and ion diffusivity have been assessed and compared to those of the related tetracyanoborate- and cyano(hydrido)borate-RTILs. The crystal structure analysis of the [BnPh3P]+ salts of [BFn(CN)4-n]- (n = 0-4), [BHn(CN)4-n]- (n = 1-3) and [BFH(CN)2]- provided experimental access to anion volumina that together with ion molecular mass, electrostatic potential, shape and chemical stability have been correlated to physicochemical properties. In addition, the cytotoxicity of the [EMIm]+-ILs and potassium or sodium salts was studied.

Azobenzene derivatives with activity against drug-resistant Candida albicans and Candida auris.

Increasing resistance against antimycotic drugs challenges anti-infective therapies today and contributes to the mortality of infections by drug-resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'-dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole-susceptible and fluconazole-resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.

An exploratory study on the effect of mechanical stress on particle formation in monoclonal antibody infusions.

Monoclonal antibody infusions (mAb-i) are administered for the treatment of various diseases. They are often transported over long distances from the compounding site to the site of administration. However, transport studies are typically carried out with the original drug product but not with compounded mAb-i. To address this gap, the impact of mechanical stress on the formation of subvisible/nanoparticles in mAb-i was investigated by dynamic light scattering and flow imaging microscopy. Different mAb-i concentrations were subjected to vibrational orbital shaking and stored at 2-8°C up to 35 days. The screening revealed that pembrolizumab and bevacizumab infusions show the highest propensity for particle formation. Especially bevacizumab at low concentrations exhibited an increase in particle formation. Because of the unknown health risks associated with the long-term application of subvisible particles (SVPs)/nanoparticles in infusion bags, stability studies carried out in the frame of licensing application procedures should also focus on SVP formation in mAb-i. In general, pharmacists should minimize the time of storage and mechanical stress during transport, especially in the case of low-concentrated mAb-i. Moreover, if siliconized syringes are used, they should be washed once with saline solution to minimize particle entry.

Chemo-Enzymatic PEGylation/POxylation of Murine Interleukin-4.

Interleukin-4 (IL-4) is a potentially interesting anti-inflammatory therapeutic, which is rapidly excreted. Therefore, serum half-life extension by polymer conjugation is desirable, which may be done by PEGylation. Here, we use PEtOx as an alternative to PEG for bioconjugate engineering. We genetically extended murine IL-4 (mIL-4) with the d-domain of insulin-like growth factor I (IGF-I), a previously identified substrate of transglutaminase (TG) Factor XIIIa (FXIIIa). Thereby, engineered mIL-4 (mIL-4-TG) became an educt for TG catalyzed C-terminal, site-directed conjugation. This was deployed to enzymatically couple an azide group containing peptide sequence to mIL-4, allowing C-terminal bioconjugation of polyethylene glycol or poly(2-ethyl-2-oxazoline). Both bioconjugates had wild-type potency and alternatively polarized macrophages.

Predicting Bile and Lipid Interaction for Drug Substances.

Predicting biopharmaceutical characteristics and food effects for drug substances may substantially leverage rational formulation outcomes. We established a bile and lipid interaction prediction model for new drug substances and further explored the model for the prediction of bile-related food effects. One hundred and forty-one drugs were categorized as bile and/or lipid interacting and noninteracting drugs using 1H nuclear magnetic resonance (NMR) spectroscopy. Quantitative structure-property relationship modeling with molecular descriptors was applied to predict a drug's interaction with bile and/or lipids. Bile interaction, for example, was indicated by two descriptors characterizing polarity and lipophilicity with a high balanced accuracy of 0.8. Furthermore, the predicted bile interaction correlated with a positive food effect. Reliable prediction of drug substance interaction with lipids required four molecular descriptors with a balanced accuracy of 0.7. These described a drug's shape, lipophilicity, aromaticity, and hydrogen bond acceptor capability. In conclusion, reliable models might be found through drug libraries characterized for bile interaction by NMR. Furthermore, there is potential for predicting bile-related positive food effects.

PEtOxylated Interferon-α2a Bioconjugates Addressing H1N1 Influenza A Virus Infection.

Influenza A viruses (IAV), including the pandemic 2009 (pdm09) H1N1 or avian influenza H5N1 virus, may advance into more pathogenic, potentially antiviral drug-resistant strains (including loss of susceptibility against oseltamivir). Such IAV strains fuel the risk of future global outbreaks, to which this study responds by re-engineering Interferon-α2a (IFN-α2a) bioconjugates into influenza therapeutics. Type-I interferons such as IFN-α2a play an essential role in influenza infection and may prevent serious disease courses. We site-specifically conjugated a genetically engineered IFN-α2a mutant to poly(2-ethyl-2-oxazoline)s (PEtOx) of different molecular weights by strain-promoted azide-alkyne cyclo-addition. The promising pharmacokinetic profile of the 25 kDa PEtOx bioconjugate in mice echoed an efficacy in IAV-infected ferrets. One intraperitoneal administration of this bioconjugate, but not the marketed IFN-α2a bioconjugate, changed the disease course similar to oseltamivir, given orally twice every study day. PEtOxylated IFN-α2a bioconjugates may expand our therapeutic arsenal against future influenza pandemics, particularly in light of rising first-line antiviral drug resistance to IAV.

Isolation and Characterization of Galloylglucoses Effective against Multidrug-Resistant Strains of Escherichia coli and Klebsiella pneumoniae.

The search for new antibiotics against multidrug-resistant (MDR), Gram-negative bacteria is crucial with respect to filling the antibiotics development pipeline, which is subject to a critical shortage of novel molecules. Screening of natural products is a promising approach for identifying antimicrobial compounds hosting a higher degree of novelty. Here, we report the isolation and characterization of four galloylglucoses active against different MDR strains of Escherichia coli and Klebsiella pneumoniae. A crude acetone extract was prepared from Paeonia officinalis Linnaeus leaves, and bioautography-guided isolation of active compounds from the extract was performed by liquid-liquid extraction, as well as open column, flash, and preparative chromatographic methods. Isolated active compounds were characterized and elucidated by a combination of spectroscopic and spectrometric techniques. In vitro antimicrobial susceptibility testing was carried out on E. coli and K. pneumoniae using 2 reference strains and 13 strains hosting a wide range of MDR phenotypes. Furthermore, in vivo antibacterial activities were assessed using Galleria mellonella larvae, and compounds 1,2,3,4,6-penta-O-galloyl-ß-d-glucose, 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-ß-d-glucose, 6-O-digalloyl-1,2,3,4-tetra-O-galloyl-ß-d-glucose, and 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-ß-d-glucose were isolated and characterized. They showed minimum inhibitory concentration (MIC) values in the range of 2-256 µg/mL across tested bacterial strains. These findings have added to the number of known galloylglucoses from P. officinalis and highlight their potential against MDR Gram-negative bacteria.

Drug-Induced Dynamics of Bile Colloids.

Bile colloids containing taurocholate and lecithin are essential for the solubilization of hydrophobic molecules including poorly water-soluble drugs such as Perphenazine. We detail the impact of Perphenazine concentrations on taurocholate/lecithin colloids using analytical ultracentrifugation, dynamic light scattering, small-angle neutron scattering, nuclear magnetic resonance spectroscopy, coarse-grained molecular dynamics simulations, and isothermal titration calorimetry. Perphenazine impacted colloidal molecular arrangement, structure, and binding thermodynamics in a concentration-dependent manner. At low concentration, Perphenazine was integrated into stable and large taurocholate/lecithin colloids and close to lecithin. Integration of Perphenazine into these colloids was exothermic. At higher Perphenazine concentration, the taurocholate/lecithin colloids had an approximately 5-fold reduction in apparent hydrodynamic size, heat release was less exothermic upon drug integration into the colloids, and Perphenazine interacted with both lecithin and taurocholate. In addition, Perphenazine induced a morphological transition from vesicles to wormlike micelles as indicated by neutron scattering. Despite these surprising colloidal dynamics, these natural colloids successfully ensured stable relative amounts of free Perphenazine throughout the entire drug concentration range tested here. Future studies are required to further detail these findings both on a molecular structural basis and in terms of in vivo relevance.

Molecular Insights into Site-Specific Interferon-α2a Bioconjugates Originated from PEG, LPG, and PEtOx.

Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a's secondary structure and only marginally reduced IFN-α2a's bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein's surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

Carbon Monoxide Exerts Functional Neuroprotection After Cardiac Arrest Using Extracorporeal Resuscitation in Pigs.

OBJECTIVES: Neurologic damage following cardiac arrest remains a major burden for modern resuscitation medicine. Cardiopulmonary resuscitation with extracorporeal circulatory support holds the potential to reduce morbidity and mortality. Furthermore, the endogenous gasotransmitter carbon monoxide attracts attention in reducing cerebral injury. We hypothesize that extracorporeal resuscitation with additional carbon monoxide application reduces neurologic damage. DESIGN: Randomized, controlled animal study. SETTING: University research laboratory. SUBJECTS: Landrace-hybrid pigs. INTERVENTIONS: In a porcine model, carbon monoxide was added using a novel extracorporeal releasing system after resuscitation from cardiac arrest. MEASUREMENTS AND MAIN RESULTS: As markers of cerebral function, neuromonitoring modalities (somatosensory-evoked potentials, cerebral oximetry, and transcranial Doppler ultrasound) were used. Histopathologic damage and molecular markers (caspase-3 activity and heme oxygenase-1 expression) were analyzed. Cerebral oximetry showed fast rise in regional oxygen saturation after carbon monoxide treatment at 0.5 hours compared with extracorporeal resuscitation alone (regional cerebral oxygen saturation, 73% ± 3% vs 52% ± 8%; p < 0.05). Median nerve somatosensory-evoked potentials showed improved activity upon carbon monoxide treatment, whereas post-cardiac arrest cerebral perfusion differences were diminished. Histopathologic damage scores were reduced compared with customary resuscitation strategies (hippocampus: sham, 0.4 ± 0.2; cardiopulmonary resuscitation, 1.7 ± 0.4; extracorporeal cardiopulmonary resuscitation, 2.3 ± 0.2; extracorporeal cardiopulmonary resuscitation with carbon monoxide application [CO-E-CPR], 0.9 ± 0.3; p < 0.05). Furthermore, ionized calcium-binding adaptor molecule 1 staining revealed reduced damage patterns upon carbon monoxide treatment. Caspase-3 activity (cardiopulmonary resuscitation, 426 ± 169 pg/mL; extracorporeal cardiopulmonary resuscitation, 240 ± 61 pg/mL; CO-E-CPR, 89 ± 26 pg/mL; p < 0.05) and heme oxygenase-1 (sham, 1 ± 0.1; cardiopulmonary resuscitation, 2.5 ± 0.4; extracorporeal cardiopulmonary resuscitation, 2.4 ± 0.2; CO-E-CPR, 1.4 ± 0.2; p < 0.05) expression were reduced after carbon monoxide exposure. CONCLUSIONS: Carbon monoxide application during extracorporeal resuscitation reduces injury patterns in neuromonitoring and decreases histopathologic cerebral damage by reducing apoptosis. This may lay the basis for further clinical translation of this highly salutary substance.

Controlling Supramolecular Structures of Drugs by Light.

Controlling physicochemical properties of light-unresponsive drugs, by light, prima facie, a paradox approach. We expanded light control by ion pairing light-unresponsive salicylate or ibuprofen to photoswitchable azobenzene counterions, thereby reversibly controlling supramolecular structures, hence the drugs' physicochemical and kinetic properties. The resulting ion pairs photoliquefied into room-temperature ionic liquids under ultraviolet light. Aqueous solutions showed trans-cis-dependent supramolecular structures under a light with wormlike aggregates decomposing into small micelles and vice versa. Light control allowed for permeation through membranes of cis-ibuprofen ion pairs within 12 h in contrast to the trans ion pairs requiring 72 h. In conclusion, azobenzene ion-pairing expands light control of physicochemical and kinetic properties to otherwise light-unresponsive drugs.

Extracorporeal resuscitation with carbon monoxide improves renal function by targeting inflammatory pathways in cardiac arrest in pigs.

Deleterious consequences like acute kidney injury frequently occur upon successful resuscitation from cardiac arrest. Extracorporeal life support is increasingly used to overcome high cardiac arrest mortality. Carbon monoxide (CO) is an endogenous gasotransmitter, capable of reducing renal injury. In our study, we hypothesized that addition of CO to extracorporeal resuscitation hampers severity of renal injury in a porcine model of cardiac arrest. Hypoxic cardiac arrest was induced in pigs. Animals were resuscitated using a conventional [cardiopulmonary resuscitation (CPR)], an extracorporeal (E-CPR), or a CO-assisted extracorporeal (CO-E-CPR) protocol. CO was applied using a membrane-controlled releasing system. Markers of renal injury were measured, and histopathological analyses were carried out. We investigated renal pathways involving inflammation as well as apoptotic cell death. No differences in serum neutrophil gelatinase-associated lipocalin (NGAL) were detected after CO treatment compared with Sham animals (Sham 71 ± 7 and CO-E-CPR 95 ± 6 ng/mL), while NGAL was increased in CPR and E-CPR groups (CPR 135 ± 11 and E-CPR 124 ± 5 ng/mL; P < 0.05). Evidence for histopathological damage was abrogated after CO application. CO increased renal heat shock protein 70 expression and reduced inducible cyclooxygenase 2 (CPR: 60 ± 8; E-CPR 56 ± 8; CO-E-CPR 31 ± 3 µg/mL; P < 0.05). Caspase 3 activity was decreased (CPR 1,469 ± 276; E-CPR 1,670 ± 225; CO-E-CPR 755 ± 83 pg/mL; P < 0.05). Furthermore, we found a reduction in renal inflammatory signaling upon CO treatment. Our data demonstrate improved renal function by extracorporeal CO treatment in a porcine model of cardiac arrest. CO reduced proinflammatory and proapoptotic signaling, characterizing beneficial aspects of a novel treatment option to overcome high mortality.

Loading-Dependent Structural Model of Polymeric Micelles Encapsulating Curcumin by Solid-State NMR Spectroscopy.

Detailed insight into the internal structure of drug-loaded polymeric micelles is scarce, but important for developing optimized delivery systems. We observed that an increase in the curcumin loading of triblock copolymers based on poly(2-oxazolines) and poly(2-oxazines) results in poorer dissolution properties. Using solid-state NMR spectroscopy and complementary tools we propose a loading-dependent structural model on the molecular level that provides an explanation for these pronounced differences. Changes in the chemical shifts and cross-peaks in 2D NMR experiments give evidence for the involvement of the hydrophobic polymer block in the curcumin coordination at low loadings, while at higher loadings an increase in the interaction with the hydrophilic polymer blocks is observed. The involvement of the hydrophilic compartment may be critical for ultrahigh-loaded polymer micelles and can help to rationalize specific polymer modifications to improve the performance of similar drug delivery systems.

Geometrical and Structural Dynamics of Imatinib within Biorelevant Colloids.

Solubilization of lipophilic drugs is essential for efficient uptake. We detail the solubilization of imatinib in simulated gastrointestinal fluids containing taurocholate (TC) and lecithin (L) and reflecting fasted versus fed states using NMR spectroscopy, X-ray diffractometry, transmission electron microscopy, and dynamic light scattering analysis. Imatinib concentration impacted colloidal geometries and molecular dynamics in a fasted state. At drug substance concentrations up to 250 µM, imatinib was mainly engulfed within the core of >110 nm in diameter vesicles. At higher drug concentrations, the colloids collapsed to <40 nm, and imatinib migrated into the shell of the micelles, mainly being associated with the lipophilic face of TC but not with L. Simulating the fed state resulted in the formation of small micelles independent of the drug concentration. Furthermore, a hydrogel was formed, effectively keeping the drug substance in an amorphous state even when stressed by drying. In conclusion, this study detailed the fascinating dynamics of colloidal structures and molecular assembly as a function of imatinib concentration in biorelevant conditions. This approach may provide a blueprint for the rational development of future pharmaceutical formulations, taking the molecular interactions with bile salts/phospholipids into account.

Investigations into neuroprotectivity, stability, and water solubility of 7-O-cinnamoylsilibinin, its hemisuccinate and dehydro derivatives.

Derivatives of the recently described potent neuroprotective 7-O-cinnamoylsilibinin ester were prepared: its hemisuccinate to improve water solubility and the dehydrosilibinin ester that was shown to form in assay media to investigate its role in overall neuroprotective effects. 7-O-Cinnamoyl-2,3-dehydrosilibinin is less neuroprotective than 7-O-cinnamoylsilibinin in a murine hippocampal cell line (HT-22) and we conclude that the dehydrosilibinin derivatives are not the actual carriers of neuroprotective properties, at least in the assay applied. Solubility of the test compounds was determined in shake-flask experiments and the ester's solubility was greatly improved by introduction of a hemisuccinate at the 23-position of silibinin. Time-stability curves in assay media were recorded. The hemisuccinate ester did not act as a prodrug to release 7-O-cinnamoylsilibinin but is the second ester bond to be cleaved. Nevertheless, it still exhibits significant neuroprotection. Therefore, its greatly increased solubility might effectively counterbalance lower in vitro neuroprotection.

Characterization of complexes between phenethylamine enantiomers and ß-cyclodextrin derivatives by capillary electrophoresis-Determination of binding constants and complex mobilities.

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte-cyclodextrin-complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different ß-cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x-reciprocal, y-reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with ß-cyclodextrin, (2-hydroxypropyl)-ß-cyclodextrin, methyl-ß-cyclodextrin and 6-O-α-maltosyl-ß-cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer-cyclodextrin-complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and ß-cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes.