Separation of terbutaline enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes.

The affinity pattern of terbutaline enantiomers towards various cyclodextrins was studied using capillary electrophoresis. The affinity pattern of terbutaline enantiomers was the same towards all studied cyclodextrins except heptakis(2-O-methyl-3,6-di-O-sulfo)-ß-CD. Nuclear magnetic resonance spectroscopy was used for understanding of fine structural mechanisms of interactions of ß-cyclodextrin and its two sulfated derivatives with the enantiomers of terbutaline. The structure of terbutaline complexes with all 3 cyclodextrins studied was different from each other. In confirmation with our earlier studies it was shown again that capillary electrophoresis represents very sensitive technique for studies of affinity patterns in cyclodextrin complexes with chiral guests. Other instrumental (e.g. NMR spectroscopy and X-ray diffraction analysis) and theoretical techniques, although very useful for obtaining the information regarding the stoichiometry, binding constants and structure of intermolecular complexes, as well as about the forces involved in selector-selectand binding and chiral recognition, may sometimes fail to properly sense those fine differences in the affinity patterns. Therefore, it is recommended to use capillary electrophoresis in order to examine correctness of affinity pattern determined for intermolecular complexes of cyclodextrins with guest molecules by other instrumental or computation techniques.

Synthesis, analytical characterization and capillary electrophoretic use of the single-isomer heptakis-(6-O-sulfobutyl)-beta-cyclodextrin.

This contribution reports the synthesis, characterization and capillary electrophoretic application of heptakis-(6-O-sulfobutyl-ether)-ß-cyclodextrin sodium salt, (6-(SB)7-ß-CD). The compound was obtained through a five-steps synthesis and it represents the first example of single-isomer sulfobutylated cyclodextrin that carries the negatively charged functions exclusively on its primary side and it is unmodified on the lower rim. The purity of each intermediate was determined by appropriate liquid chromatographic methods, while the isomeric purity of the final product was established by an ad-hoc developed HPLC method based on a CD-Screen-IEC column. The structural identification of 6-(SB)7-ß-CD was carried out by 1D, 2D NMR spectroscopy and ESI-MS. The chiral separation ability of 6-(SB)7-ß-CD was studied by chiral capillary electrophoresis using the single-isomer host as a background electrolyte additive to separate the enantiomers of a representative set of pharmacologically significant model compounds such as verapamil, dapoxetine, ondansetron, propranolol, atenolol, metoprolol, carvedilol, terbutaline, amlodipine and tadalafil. The enantiomer migration order and the effects of the selector concentration on the enantiorecognition properties were investigated. NMR spectroscopy was applied to deepen and further confirm the host-guest interactions and in the case of the model compound dapoxetine a potential representation for the supramolecular assembly was developed based on the dataset collected by the extensive 2D NMR analysis. This single-isomer chiral selector offers a new alternative to the widely applied randomly sulfobutylated- and sulfated-beta-cylodextrins as well as to the single-isomer sulfated and carboxymethylated derivatives in chiral separations.

Characterization of a single-isomer carboxymethyl-beta-cyclodextrin in chiral capillary electrophoresis.

In this work, the synthesis, characterization, and chiral capillary electrophoretic study of heptakis-(2,3-di-O-methyl-6-O-carboxymethyl)-ß-CD (HDMCM), a single-isomer carboxymethylated CD, are presented. The pH-dependent and selector concentration-dependent enantiorecognition properties of HDMCM were investigated and discussed herein. The enantioseparation was assessed applying a structurally diverse set of noncharged, basic, and zwitterionic racemates. The increase in the selector concentration and gross negative charge of HDMCM improved the enantioseparation that could be observed in the majority of the cases. HDMCM was also successfully applied as BGE additive in NACE using a methanol-based system in order to prove the separation selectivity features and to highlight the broad applicability of HDMCM. Over 25 racemates showed partial or baseline separation with HDMCM under the conditions investigated, among which optimal enantiomer migration order was found for the four stereoisomers of tadalafil, tapentadol, and dapoxetine, offering the possibility of a chiral CE method development for chiral purity profiling of these drugs.

Separation of enilconazole enantiomers in capillary electrophoresis with cyclodextrin-type chiral selectors and investigation of structure of selector-selectand complexes by using nuclear magnetic resonance spectroscopy.

In the present study, the enantiomer migration order (EMO) of enilconazole in the presence of various cyclodextrins (CDs) was investigated by capillary electrophoresis (CE). Opposite EMO of enilconazole were observed when ß-CD or the sulfated heptakis(2-O-methyl-3,6-di-O-sulfo)-ß-CD (HMDS-ß-CD) was used as the chiral selectors. Nuclear magnetic resonance (NMR) spectroscopy was used to study the mechanism of chiral recognition between enilconazole enantiomers and those two cyclodextrins. On the basis of rotating frame nuclear Overhauser (ROESY) experiments, the structure of an inclusion complex between enilconazole and ß-CD was derived, in which (+)-enilconazole seemed to form a tighter complex than the (-)-enantiomer. This correlates well with the migration order of enilconazole enantiomers observed in CE. No evidence of complexation between enilconazole and HMDS-ß-CD could be gathered due to lack of intermolecular nuclear Overhauser effect (NOE). Most likely the interaction between enilconazole and HMDS-ß-CD leads to formation of a shallow external complex that is sufficient for separation of enantiomers in CE but cannot be evidenced based on ROESY experiment. Thus, in this particular case CE documents the presence of intermolecular interactions which are at least very difficult to be evidenced by other instrumental techniques.

Single-isomer carboxymethyl-γ-cyclodextrin as chiral resolving agent for capillary electrophoresis.

Herein we report on the synthesis, characterization and the novel capillary electrophoretic use of octakis-(2,3-di-O-methyl-6-O-carboxymethyl)-γ-cyclodextrin sodium salt (ODMCM). ODMCM is the first single-isomer carboxymethyl-γ-cyclodextrin that is fully methylated on its secondary side and carries ionizable carboxymethyl functions on its primary side. ODMCM was prepared with high isomeric purity through a four-step synthetic procedure. The purity of each intermediate was characterized by appropriate chromatographic methods, while the isomeric purity of the carboxymethylated product was determined by an HPLC method using a CD-Screen-IEC column and by a capillary electrophoretic method using indirect UV detection, as well. The structural identification of the ODMCM was carried out by 1D, 2D NMR spectroscopy and ESI-MS. The acid-base characterization of the chiral selector was carried out by 1H NMR-pH titration. The chiral separation ability of the synthesized selector was studied by chiral capillary electrophoresis. ODMCM was used as a background electrolyte additive to separate enantiomers of representative pharmacologically significant model molecules such as propranolol, citalopram, ketamine, tapentadol and dapoxetine. The effects of the selector concentration and the pH of the background electrolyte on the enantiorecognition properties were investigated. 1H NMR spectroscopy was further applied to get deeper insight of the host-guest inclusion complex formation. The pH-dependent enantioselectivity of this new single-isomer chiral selector was demonstrated by chiral capillary electrophoresis and 1H NMR spectroscopy.

"Back to the Future": A New Look at Hydroxypropyl Beta-Cyclodextrins.

Since the discovery about 30 years ago (2-hydroxypropyl) beta-cyclodextrin, a highly soluble derivative of beta-cyclodextrin, has become an approved excipient of drug formulations included both in the United States and European Pharmacopoeias. It is recommended to use as solubilizer and stabilizer for oral and parenteral formulations. Recently, its pharmacological activity has been recognized in various diseases. The increasing applications require a closer look to the structure-activity relationship. As (2-hydroxypropyl) beta-cyclodextrin (HPBCD) is always a mixture of isomers with various degrees and pattern of hydroxypropylation, no wonder that the products of different manufacturers are often different. Several HPBCDs were compared applying a battery of analytical tools including thin layer chromatography, high performance liquid chromatography (HPLC), HPLC-mass spectrometry (MS), and matrix-assisted laser desorption MS. We studied how the average degree of substitution affects the aggregation behavior, the toxicity, and the solubilizing effect on poorly soluble drugs. We found that the products with low average degree of substitution are more prone to aggregation. The samples studied are nontoxic to Caco-2 cells and have low hemolytic activity. The solubility enhancement of poorly soluble drugs decreases or increases with increasing degree of substitution or shows a maximum curve depending on the properties of the guest.

Analytical characterization of cyclodextrins: History, official methods and recommended new techniques.

The main goal of this review is to provide a comprehensive overview on the methods used for analysis of cyclodextrins (CDs) and CD-derivatives. The paper intends to act as a guide for the readers in looking around the classical and modern instrumental analytical methods suitable for identification, characterization and determination of CDs themselves, CDs in finished products or even in biological samples. At present, in the European and United States Pharmacopoeias, the three parent CDs and two synthetic derivatives, namely the (2-hydroxypropyl)-beta-CD and sulfobutylether-beta-CD Na salt are official. Besides these modified CDs, two other derivatives are approved as excipients in human pharmaceutical products: the (2-hydroxypropyl)-gamma-CD and the randomly methylated-beta-CD. Although most of the official analysis methods in the pharmacopoeias have been well used for decades, new aspects of the functional excipient CD characterization suggest a need to revisit compendial methods. Comparison of strengths and weaknesses of current official methods with new improved techniques intends to help analysts to decide on changing traditional analytical methods with improved new ones. This review also deals with the analytical aspects of the first single isomer CD derivative approved as a drug active (Sugammadex/Bridion®) as well as analytical considerations of using CDs themselves as active pharmaceutical ingredients. Stability-indicating instrumental methods suitable to adequately follow chemical- and enzymatic degradation of CDs will also be discussed. Challenges in the determination of CDs in different biological matrices will be illustrated on real pharmaco- and toxicokinetic studies of CD-enabled drug formulations.

Efficacy and ototoxicity of different cyclodextrins in Niemann-Pick C disease.

OBJECTIVE: Niemann-Pick type C (NPC) disease is a fatal, neurodegenerative, lysosomal storage disorder characterized by intracellular accumulation of unesterified cholesterol (UC) and other lipids. While its mechanism of action remains unresolved, administration of 2-hydroxypropyl-ß-cyclodextrin (HPßCD) has provided the greatest disease amelioration in animal models but is ototoxic. We evaluated other cyclodextrins (CDs) for treatment outcome and chemical interaction with disease-relevant substrates that could pertain to mechanism. METHODS: NPC disease mice treated for 2 weeks with nine different CDs were evaluated for UC, and GM2 and GM3 ganglioside accumulation using immunohisto/cytochemical and biochemical assays. Auditory brainstem responses were determined in wild-type mice administered CDs. CD complexation with UC, gangliosides, and other lipids was quantified. RESULTS: Four HPßCDs varying in degrees of substitution, including one currently in clinical trial, showed equivalent storage reduction, while other CDs showed significant differences in relative ototoxicity and efficacy, with reductions similar for the brain and liver. Importantly, HPγCD and two sulfobutylether-CDs showed efficacy with reduced ototoxicity. Complexation studies showed: incomplete correlation between CD efficacy and UC solubilization; an inverse correlation for ganglioside complexation; substantial interaction with several relevant lipids; and association between undesirable increases of UC storage in Kupffer cells and UC solubilization. INTERPRETATION: CDs other than HPßCD identified here may provide disease amelioration without ototoxicity and merit long-term treatment studies. While direct interactions of CD-UC are thought central to the mechanism of correction, the data show that this does not strictly correlate with complexation ability and suggest interactions with other NPC disease-relevant substrates should be considered.

Cationic permethylated 6-monoamino-6-monodeoxy-ß-cyclodextrin as chiral selector of dansylated amino acids in capillary electrophoresis.

The resolution power of permethylated 6-monoamino-6-monodeoxy-ßCD (PMMABCD) - a single isomer, cationic CD derivative - developed previously for chiral analyses in capillary electrophoresis was further studied here. Dansylated amino acids (Dns-AA) were chosen as amphoteric chiral model compounds. Changes in the resolutions of Dns-AAs by varying pH and selector concentrations were investigated and correlated with their structures and chemical properties (isoelectric point and lipophilicity). Maximal resolutions could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of the selector. Baseline or substantially better resolution for 8 pairs of these Dns-AAs could be achieved. Low CD concentration was enough for the separation of the most apolar Dns-AAs. Chiral discrimination ability of PMMABCD was demonstrated by the separation of an artificial mixture of 8 Dns-AA pairs.

Methyl-beta-cyclodextrins: the role of number and types of substituents in solubilizing power.

Methylated cyclodextrins (CDs) are effective solubilizers of poorly soluble organic compounds. In this work, we compared various methylated ß-CDs concerning their structure characterized by nuclear magnetic resonance spectroscopy, composition analyzed by HPLC and solubilizing capability by using model compounds such as cholesterol, fatty acids, furosemide, tamoxifen, and amiodarone. All the commercially available methylated ß-CDs are mixtures of various isomers and homologues except trimethyl ß-CD. The effects of the degree of methylation, the composition, as well as the influence of further derivatization with ionic groups were studied. The number of methyl groups in a CD ring should be around 14 to get the highest solubility for the included guest molecules. Although the distribution of isomers and related compounds has hardly any effect at constant degree of substitution, the introduction of amino and succinyl moieties on the CD ring adds ionic interactions to the hydrophobic interactions of the inclusion complex formation, which might result in synergic effect in solubilization.

Cyclodextrins in analytical chemistry: host-guest type molecular recognition.

Cyclodextrins are utilized in many diverse fields of analytical chemistry, due to their propensity to form reversible inclusion complexes and recognize analytes selectively. This Feature shows how these nanocavities can serve analysts in sample preparation, sensitivity and selectivity improvement, enantio-separation, creating single-molecule sensors, and automatizing DNA sequencing.

Structure and stability of warfarin-sodium inclusion complexes formed with permethylated monoamino-ß-cyclodextrin.

Inclusion complexes of warfarin enantiomers with permethylated monoamino-ß-cyclodextrin (PMMABCD) were characterized using CE and (1)H NMR spectroscopy in aqueous solution. These techniques gave complementary information on the stability and the structure of the diastereomeric host-guest inclusion complexes. The stability constants were determined from CE experiments in a wide pH range. Change in the migration order on the variation of the pH was observed. (1)H NMR assignments have been established for the seven non-equivalent carbohydrate units of the host in the complex at pH 7-9. Specific H-H distance restraints were obtained from NOESY experiments and were introduced into molecular modeling to establish the geometry of the inclusion complexes. It was found that the open side chain warfarin enters the cavity from the primary side of the CD. The orientation of the coumarin ring within the cavity has the same preference for the two warfarin enantiomers owing to an ionic interaction with the amino group of the CD. Accordingly, enantioselectivity at pH 8.5 arises from the difference in the CH/π interactions between warfarin aromatics and the manifold of CH groups of the CD.

Comparison of separation performances of novel ß-cyclodextrin-based chiral stationary phases in high-performance liquid chromatographic enantioseparation.

Three ß-cyclodextrin-based chiral stationary phases were developed applying novel bonding chemistry. The separation performances of ß-cyclodextrin, (R,S)-2-hydroxypropyl-ß-cyclodextrin, and permethyl-ß-cyclodextrin-based CSPs were compared in the resolution of structurally divergent analytes, such as coumarins, dansyl amino acids, and propionic acid derivatives. Separations were carried out in reversed phase mode applying 0.1% triethylammonium phosphate (pH 3.5)/MeOH mobile phase systems in different compositions. Of the three novel CSPs the permethyl-ß-cyclodextrin bonded phase proved to be the most effective one for the enantioseparation of investigated analytes.

Diverse associations in the ternary systems of ß-cyclodextrin, simple carbohydrates and phenyl derivatives of inorganic oxoacids.

Complex formation reactions of phenylboronic, phenylphosphonic, phenylarsonic and 4-aminophenyl arsonic acids with ß-cyclodextrin (cycloheptaamylose, ß-CD) and some simple carbohydrates (mannitol, sorbitol, glucose) have been studied using spectrophotometric, potentiometric methods and solubility measurements, supplemented with HPLC and IR analyses of the solid samples. Equilibrium constants have been determined at ionic strength of 0.2M (NaCl) and 25°C. ß-CD forms the most stable complexes with the neutral, undissociated forms of the acids, the stability constants are as follows: phenylboronic acid: 320 ± 36, phenylphosphonic acid: 108 ± 25, phenylarsonic acid: 97 ± 4 and 4-aminophenyl arsonic acid: 107 ± 10. The stability constants for the ß-CD-complexes of the ionic forms are much lower. Ternary complexes of low stability could be detected in the case of phenylphosphonic acid and sorbitol with the undissociated form and with glucose and the dianion. In more concentrated solutions phenylboronic acid forms insoluble complexes with mannitol, sorbitol and ß-CD. The solid phases obtained in the ternary systems are predominantly mixtures of ester type 3:1 complexes with the carbohydrate and 1:1 inclusion complex with the ß-CD. No significant interaction has been found with glucose. The phenomena can be explained by the differences in the structures of the components and by the changes in the H-bonding network of ß-CD on the complex formation.

Enantiomeric separation of antimalarial drugs by capillary electrophoresis using neutral and negatively charged cyclodextrins.

Capillary electrophoresis (CE) methods for chiral resolution of five antimalarial drugs (primaquine, tafenoquine, mefloquine, chloroquine and quinacrine) were developed by using a wide selection of neutral and anionic cyclodextrin (CD) derivatives. The use of sulfobutyl-ß-CD and carboxymethyl-ß-CD (CMBCD) resulted in good resolution of quinacrine and tafenoquine, respectively. New results are presented for resolutions of chloroquine and mefloquine. Application of carboxyalkyl- and sulfobutyl-CD derivatives provided improved resolution for primaquine. The impurity in primaquine sample detected by CE was identified as quinocide by MS and NMR. CMBCD provided not only the best separation of primaquine from quinocide but also the simultaneous complete resolution of both compounds.

Cyclodextrin KnowledgeBase a web-based service managing CD-ligand complexation data.

Cyclodextrins are cyclic oligosaccharides that are able to form water-soluble inclusion complexes with small molecules. Because of their complexing ability, they are widely applied in food, pharmaceutical and chemical industries. In this paper we describe the development of a free web-service, Cyclodextrin KnowledgeBase: ( http://www.cyclodextrin.net ). The database contains four modules: the Publication, Interaction, Chirality and Analysis Modules. In the Publication Module, almost 50,000 publication details are collected that can be retrieved by text search. In the Interaction and Chirality Modules relevant literature data on cyclodextrin complexation and chiral recognition are collected that can be retrieved by both text and structural searches. Moreover, in the Analysis Module, the geometries of small molecule-cyclodextrin complexes can be predicted using molecular docking tools in order to explore the structures and interaction energies of the inclusion complexes. Complex geometry prediction is made possible by the built-in database of 95 cyclodextrin derivatives, where the 3D structures as well as the partial charges are calculated and stored for further utilization. The use of the database is demonstrated by several examples.

Separation of cis-beta-lactam enantiomers by capillary electrophoresis using cyclodextrin derivatives.

Chiral separation of 19 pairs of cis-beta-lactam (BL) stereoisomers of pharmacological importance was examined by capillary electrophoresis using cyclodextrin (CD) derivatives. In order to select the most effective conditions separating the highest number of stereoisomers of BLs, single carboxymethyl alpha-, beta- and gamma-, as well as sulfobutyl beta-CD derivatives were applied. Additionally, carboxymethyl and sulfobutyl beta-CD derivatives complemented with neutral beta-CD derivatives as dual CD systems were tested. Both the composition and concentration of applied selectors and the pH of background electrolyte were selected. In single systems the structural characteristics of BLs and the complex forming affinity were correlated. Most BLs provided optimal complexation with beta-CD derivatives. In conclusion, the efficiency of combining sulfobutyl-beta-CD and permethylated beta-CD was superior to other single and dual CD systems applied. This method successfully separated each pair of stereoisomers investigated.

Chiral separation by a monofunctionalized cyclodextrin derivative: from selector to permethyl-beta-cyclodextrin bonded stationary phase.

Preparation of (6-monoureido-6-monodeoxy) permethylated beta-cyclodextrin bonded chiral stationary phase from permethylated 6-monoamino-6-monodeoxy-beta-cyclodextrin is described. The optimized chiral stationary phase was evaluated by using HPLC separation of racemates of coumarin derivatives. Column characterization was performed by solid-state (13)C, (15)N, (29)Si NMR using cross-polarization at the magic angle spinning. The development process was supported by CE experiments where the complex formation between cyclodextrins and warfarin was investigated. The results demonstrate good enantio-discrimination for coumarin derivatives.

Aggregation of cyclodextrins as an important factor to determine their complexation behavior.

Here, we report a study on the complexation behavior of carotenoids with cyclodextrins (CDs) using solubility experiments and molecular-modelling methods. Carotenoids are an important group of naturally occurring dyes found in vegetables and fruits. Their antioxidant property has initiated investigations on their possible use as drugs. However, carotenoids are lipophilic molecules with very little inherent aqueous solubility. Cyclodextrin complexation has been widely used in order to increase the potential applications of hydrophobic compounds. Thus, the aim of our investigation was to design carotenoids with enhanced water solubility by cyclodextrin complexation. Molecular modelling of carotenoid-cyclodextrin complexes with a 1 : 1 stoichiometry successfully explained the experimentally observed capability of beta-cyclodextrins (beta-CDs) to form complexes with carotenoids as opposed to alpha-cyclodextrins (alpha-CDs) and gamma-cyclodextrins (gamma-CDs). Furthermore, molecular-dynamics calculations revealed that the aggregation properties of CD derivatives significantly influence their complexation behavior. Our docking calculations showed that RAMEB (random methylated beta-CD) is the beta-CD derivative that possesses the lowest tendency to aggregate. Solubility experiments yielded the same results, namely, RAMEB complexes possess the best water solubility. Our results showed that complexation of a ligand not buried inside of the CD cavity is dependent on two factors: i) the geometry of the inclusion part of the complex; ii) the self-aggregation property of the CD itself. The lower affinity the CDs possess for self-aggregation, the more likely are they involved in interactions with carotenoids. These results suggest that self-aggregation of CDs should be considered as an important parameter determining complexation in general.

High-performance liquid chromatographic determination of 2-hydroxypropyl-gamma-cyclodextrin in different biological fluids based on cyclodextrin enhanced fluorescence.

A high-performance size exclusion chromatographic method with analyte enhanced fluorescence detection is described for the analysis of 2-hydroxypropyl-gamma-cyclodextrin (HPGCD) in different biological fluids. The principle of detection was the in situ complexation of 8-anilinonaphthalene-1-sulfonic acid (ANS) by HPGCD. When HPGCD eluted from the column the increased fluorescence was measured at excitation and emission wavelengths of 270 and 512 nm, respectively. Solid-phase extraction cleanup and concentration of samples resulted in higher than 78% recovery of HPGCD for each of the studied biological fluids. Some important details of the method development as well as the validation of the method for rabbit plasma, rabbit aqueous humour, monkey plasma and monkey urine are given. The limits of quantification varied between 1 and 10 nmol/ml (correspond to 1.5-15 microg/ml) depending on the biological matrix used. The method was successfully adapted in another laboratory proving that HPGCD had not absorbed into aqueous humour and plasma after topical application of HPGCD containing eye drop in rabbits.