Anti-Inflammatory Activity of Pyrazolo[1,5-a]quinazolines.

Chronic inflammation contributes to a number of diseases. Therefore, control of the inflammatory response is an important therapeutic goal. To identify novel anti-inflammatory compounds, we synthesized and screened a library of 80 pyrazolo[1,5-a]quinazoline compounds and related derivatives. Screening of these compounds for their ability to inhibit lipopolysaccharide (LPS)-induced nuclear factor κB (NF-κB) transcriptional activity in human THP-1Blue monocytic cells identified 13 compounds with anti-inflammatory activity (IC50 < 50 µM) in a cell-based test system, with two of the most potent being compounds 13i (5-[(4-sulfamoylbenzyl)oxy]pyrazolo[1,5-a]quinazoline-3-carboxamide) and 16 (5-[(4-(methylsulfinyl)benzyloxy]pyrazolo[1,5-a]quinazoline-3-carboxamide). Pharmacophore mapping of potential targets predicted that 13i and 16 may be ligands for three mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2), p38α, and c-Jun N-terminal kinase 3 (JNK3). Indeed, molecular modeling supported that these compounds could effectively bind to ERK2, p38α, and JNK3, with the highest complementarity to JNK3. The key residues of JNK3 important for this binding were identified. Moreover, compounds 13i and 16 exhibited micromolar binding affinities for JNK1, JNK2, and JNK3. Thus, our results demonstrate the potential for developing lead anti-inflammatory drugs based on the pyrazolo[1,5-a]quinazoline and related scaffolds that are targeted toward MAPKs.

Design, Synthesis and Pharmacological Evaluation of New Quinoline-Based Panx-1 Channel Blockers.

Pannexins are an interesting new target in medicinal chemistry, as they are involved in many pathologies such as epilepsy, ischemic stroke, cancer and Parkinson's disease, as well as in neuropathic pain. They are a family of membrane channel proteins consisting of three members, Panx-1, Panx-2 and Panx-3, and are expressed in vertebrates. In the present study, as a continuation of our research in this field, we report the design, synthesis and pharmacological evaluation of new quinoline-based Panx-1 blockers. The most relevant compounds 6f and 6g show an IC50 = 3 and 1.5 µM, respectively, and are selective Panx-1 blockers. Finally, chemical stability, molecular modelling and X-ray crystallography studies have been performed providing useful information for the realization of the project.

4,5-Dihydro-5-Oxo-Pyrazolo[1,5-a]Thieno[2,3-c]Pyrimidine: A Novel Scaffold Containing Thiophene Ring. Chemical Reactivity and In Silico Studies to Predict the Profile to GABAA Receptor Subtype.

The isosteric replacement of the benzene with thiophene ring is a chemical modification widely applied in medicinal chemistry. Several drugs containing the thiophene ring are marketed for treating various pathologies (osteoporosis, peripheral artery disorder, psychosis, anxiety and convulsion). Taking into account this evidence and as a continuation of our study in the GABAA receptor modulators field, we designed and synthesized new compounds containing the thiophene ring with 4,5-dihydro-5-oxo-pyrazolo[1,5-a]thieno[2,3-c]pyrimidine and pyrazolo[1,5-a]thieno[2,3-c] pyrimidine scaffold. Moreover, these cores, never reported in the literature, are isosteres of pyrazolo[1,5-a]quinazolines (PQ), previously published by us as GABAAR subtype ligands. We introduced in the new scaffold those functions and groups (esters, ketones, alpha/beta-thiophene) that in our PQ derivatives were responsible for the activity, and at the same time, we have extensively investigated the reactivity of the new nucleus regarding the alkylation, reduction, halogenation and hydrolyses. On the six final designed compounds (12c-f, 22a,b) molecular docking and dynamic simulation studies have been performed. The analysis of dynamic simulation, applying our reported model 'Proximity Frequencies', collocates with high probability 12c, 22b, in the agonist class towards α1ß2γ2-GABAAR.

GABAA Receptor Modulators with a Pyrazolo[1,5-a]quinazoline Core: Synthesis, Molecular Modelling Studies and Electrophysiological Assays.

As a continuation of our study in the GABAA receptor modulators field, we report the design and synthesis of new 8-chloropyrazolo[1,5-a]quinazoline derivatives. Molecular docking studies and the evaluation of the 'Proximity Frequencies' (exploiting our reported model) were performed on all the final compounds (3, 4, 6a-c, 7a,b, 8, 9, 12a-c, 13a,b, 14-19) to predict their profile on the α1ß2γ2-GABAAR subtype. Furthermore, to verify whether the information coming from this virtual model was valid and, at the same time, to complete the study on this series, we evaluated the effects of compounds (1-100 µM) on the modulation of GABAA receptor function through electrophysiological techniques on recombinant α1ß2γ2L-GABAA receptors expressed in Xenopus laevis oocytes. The matching between the virtual prediction and the electrophysiological tests makes our model a useful tool for the study of GABAA receptor modulators.

New Panx-1 Blockers: Synthesis, Biological Evaluation and Molecular Dynamic Studies.

The channel protein Panx-1 is involved in some pathologies, such as epilepsy, ischemic stroke, cancer and Parkinson's disease, as well as in neuropathic pain. These observations make Panx-1 an interesting biological target. We previously published some potent indole derivatives as Panx-1 blockers, and as continuation of the research in this field we report here the studies on additional chemical scaffolds, naphthalene and pyrazole, appropriately substituted with those functions that gave the best results as in our indole series (sulphonamide functions and one/two carboxylic groups) and in Panx-1 blockers reported in the literature (sulphonic acid). Compounds 4 and 13, the latter being an analogue of the drug Probenecid, are the most potent Panx-1 blockers obtained in this study, with I = 97% and I = 93.7% at 50 µM, respectively. Both compounds, tested in a mouse model of oxaliplatin-induced neuropathic pain, showed a similar anti-hypersensitivity profile and are able to significantly increase the mouse pain threshold 45 min after the injection of the doses of 1 nmol and 3 nmol. Finally, the molecular dynamic studies and the PCA analysis have made it possible to identify a discriminating factor able to separate active compounds from inactive ones.

'Proximity frequencies' a new parameter to evaluate the profile of GABAAR modulators.

We reported the synthesis of new 8-methoxypyrazolo[1,5-a]quinazolines bearing an amide fragment at the 3-position. The final compounds, as aromatic (2a-i) and 4,5-dihydro derivatives (3a-i), have been evaluated in vitrofor their ability to modulate the chlorine current on recombinant GABAA receptors of the α1ß2γ2L type (expressed in frog oocytes of the Xenopus laevis species). From electrophysiological test two groups of compounds emerged: positive modulators agonist (2e, h, i and 3e, h) and null modulators antagonist (2a, b, d, f, g and 3a-d, f, g) of GABAA subtype receptor. Using a set of compounds (new derivatives, known products and GABAA subtype receptor ligands from our library) we identify the amino acids at the α+/γ- interface, which could be involved in the agonist or antagonist profile, using the 'Proximity Frequencies', namely the frequencies with which a ligand intercepts two or more binding-site amino acids during the molecular dynamic simulation. The linear discriminant analysis (LDA) evidences that the combination of amino acids αVAL203- γTHR142 and αTYR 160- γTYR 58 allowed to collocate 70.6% of agonists and 72.7% of antagonists in their respective class.

Pyrazolo[1,5-a]quinazoline scaffold as 5-deaza analogue of pyrazolo[5,1-c][1,2,4]benzotriazine system: synthesis of new derivatives, biological activity on GABAA receptor subtype and molecular dynamic study.

To investigate the binding affinity of GABAA receptor subtype, new pyrazolo [1,5-a]quinazolines were designed, synthesized, and in vitro evaluated. These compounds, 5-deaza analogues of pyrazolo[5,1-c][1,2,4]benzotriazine derivatives which were already studied in our research group, permit us to evaluate the relevance of the nitrogen or the oxygen atom at 5-position of the tricyclic scaffold. Molecular dynamic study was done on a set of the new and known ligands to rationalize and to explain the lack of affinity on the 4- or 5-substituted new derivative. In fact, from biological results, it can be found that the only 5-unsubstituted new derivative, compound 15, has receptor recognition (Ki = 834.7 nM).

Substituted piperazines as nootropic agents: 2- or 3-phenyl derivatives structurally related to the cognition-enhancer DM235.

A series of 2-phenyl- or 3-phenyl piperazines, structurally related to DM235 and DM232, two potent nootropic agents, have been prepared and tested in the mouse passive-avoidance test, to assess their ability to revert scopolamine-induced amnesia. Although the newly synthesized molecules were less potent than the parent compounds, some useful information has been obtained from structure-activity relationships. A small but significant enantioselectivity has been found for the most potent compound 5a.

Design and solid phase synthesis of new DOTA conjugated (+)-biotin dimers planned to develop molecular weight-tuned avidin oligomers.

Chemical modifications of the biotin carrier in pretargeted avidin­biotin radionuclide therapy may be of paramount importance for tuning the amount of the radioactivity delivered to cancer cells by labelled biotins. We report here the synthesis of a collection of new synthetic DOTA-constructs bearing two (+)-biotin molecules (bis-biotins), designed for the creation of multimeric Av units (tetramers) bonded to the antibody. All the syntheses were carried out following the solid phase strategy and growing the molecules on a Rink Amide resin. The biotin heads are connected through spacers containing PEG or non-PEG residues. Molecular modelling calculations suggested that the Av cross-linking ability of the bis-biotins depends mainly on the spacers length, with the best results being expected for arms affording distances in the range of 10­25 Å between the biotin carboxylate atoms, in the fully extended conformation. SEC-HPLC MALLS analysis of the products of our Av/bis-biotin reaction mixtures have confirmed this hypothesis. The bis-biotin 16, where the non-PEG linker ensured a distance of 26.7 Å between the biotin moieties, gave about 50% of Av oligomers while the shorter analogue 18 (19.5 Å) afforded 100% of an Av polymer containing about 21 protein units. Remarkably, the solubility of both the bis-biotins, i.e.16 and 18, in aqueous solutions was good and they showed excellent stability against the action of peptidases.

HS-SPME-GC-MS and Chemometrics for the Quality Control and Clustering of Monovarietal Extra Virgin Olive Oil: A 3-Year Study on Terpenes and Pentene Dimers of Italian Cultivars.

Terpenes and pentene dimers are less studied volatile organic compounds (VOCs) but are associated with specific features of extra virgin olive oils (EVOOs). This study aimed to analyze mono- and sesquiterpenes and pentene dimers of Italian monovarietal EVOOs over 3 years (14 cultivars, 225 samples). A head space-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method recently validated was used for terpene and pentene dimer quantitation. The quantitative data collected were used for both the characterization and clustering of the cultivars. Sesquiterpenes were the molecules that most characterized the different cultivars, ranging from 3.908 to 38.215 mg/kg; different groups of cultivars were characterized by different groups of sesquiterpenes. Pentene dimers (1.336 and 3.860 mg/kg) and monoterpenes (0.430 and 1.794 mg/kg) showed much lower contents and variability among cultivars. The application of Kruskal-Wallis test-PCA-LDA-HCA to the experimental data allowed defining 4 clusters of cultivars and building a predictive model to classify the samples (94.3% correct classification). The model was further tested on 33 EVOOs, correctly classifying 91% of them.

Composition of discarded Sicilian fruits of Opuntia ficus indica L.: Phenolic content, mineral profile and antioxidant activity in peel, seeds and whole fruit.

Sicily (Italy) is the second producer of Opuntia ficus-indica (OFI) fruits after Mexico. To date, huge quantities of fruit are discarded during the selection for the fresh market, generating a large amount of by-product to be valorized. This study aimed to investigate on the composition of OFI discarded fruits from the main Sicilian productive areas, over two harvesting periods. Peel, seeds and whole fruit samples were characterized in terms of minerals and phenolic compounds through ICP-OES and HPLC-DAD-MS. Potassium, calcium and magnesium were the most abundant elements and peel samples showed the highest values. Seventeen phenolic compounds were detected in peel and whole fruit, including flavonoids, phenylpyruvic and hydroxycinnamic acids, while only phenolic acids were found in the seeds. A multivariate chemometric approach highlighted a correlation between the mineral and phenolic content and the different parts of the fruit as well as a significant influence of productive area.

4-Heteroaryl Substituted Amino-3,5-Dicyanopyridines as New Adenosine Receptor Ligands: Novel Insights on Structure-Activity Relationships and Perspectives.

A new set of amino-3,5-dicyanopyridines was synthesized and biologically evaluated at the adenosine receptors (ARs). This chemical class is particularly versatile, as small structural modifications can influence not only affinity and selectivity, but also the pharmacological profile. Thus, in order to deepen the structure-activity relationships (SARs) of this series, different substituents were evaluated at the diverse positions on the dicyanopyridine scaffold. In general, the herein reported compounds show nanomolar binding affinity and interact better with both the human (h) A1 and A2A ARs than with the other subtypes. Docking studies at hAR structure were performed to rationalize the observed affinity data. Of interest are compounds 1 and 5, which can be considered as pan ligands as binding all the ARs with comparable nanomolar binding affinity (A1AR: 1, Ki = 9.63 nM; 5, Ki = 2.50 nM; A2AAR: 1, Ki = 21 nM; 5, Ki = 24 nM; A3AR: 1, Ki = 52 nM; 5, Ki = 25 nM; A2BAR: 1, EC50 = 1.4 nM; 5, EC50 = 1.12 nM). Moreover, these compounds showed a partial agonist profile at all the ARs. This combined AR partial agonist activity could lead us to hypothesize a potential effect in the repair process of damaged tissue that would be beneficial in both wound healing and remodeling.

Evaluation of the separation mechanism of electrokinetic chromatography with a microemulsion and cyclodextrins using NMR and molecular modeling.

Electrokinetic chromatography (EKC) allows the separation of closely related substances by the detection of fine effects in analyte-separation system interactions. With the goal of understanding the fine effects involved in separation using a dual cyclodextrin-microemulsion EKC system, an integrated study of NMR and molecular modeling was carried out. The above dual cyclodextrin-microemulsion system was previously used in the separation of clemastine and its related substances and was prepared by the addition of methyl-ß-cyclodextrin (MßCD) and heptakis(2,6-di-O-methyl)-ß-cyclodextrin (DMßCD) to an oil-in-water microemulsion. The use of DMßCD was shown to be essential in the separation of clemastine from one of its related substance (I(B) ). A molecular modeling study allowed the different affinities of clemastine and I(B) for the two cyclodextrins to be explained. Furthermore, rotating-frame Overhauser effect spectroscopy NMR experiments clearly indicated that besides the primary pseudostationary phase, namely the ionic microemulsion, cyclodextrins acted as a secondary pseudostationary phase. In addition, it was shown that inclusion complexation of sodium dodecyl sulfate (SDS) monomers into the cyclodextrins cavity occurs; differently, the oil (n-heptane) used in the preparation of microemulsion system resulted to be not included into the macrocycle cavity. These experimental results were supported by molecular modeling, which highlighted the preferential inclusion of SDS into DMßCD. On the basis of these results, it was confirmed that, besides its primary role as the ionic carrier in EKC, SDS is involved in inclusion equilibria toward CDs, which can be effective in increasing the system selectivity.

New 3-, 8-disubstituted pyrazolo[5,1-c][1,2,4]benzotriazines useful for studying the interaction with the HBp-3 area (hydrogen bond point area) in the benzodiazepine site on the γ-aminobutyric acid type A (GABAA) receptor.

The pharmacophoric model using ADLR procedure, based on pyrazolo[5,1-c][1,2,4]benzotriazine system, studied in our laboratory, allowed us to identify the essential interaction points (HBp-1, HBp-2, and Lp-1) and the important areas for affinity modulation (HBp-3 and Lp-2) for binding recognition at benzodiazepine (Bzs) site of GABA(A) receptors (GABA(A)-Rs). In this work ADLR method is used to rationalize the affinity data of 23 new compounds and to improve the knowledge on HBp-3 area, hydrogen bond area. Among these new compounds emerge the pyrrolo derivatives (18, 25, 28, 34, and 37) for their good affinity value (14.9>K(i)(nM)>63.0). In the orientations proposed by ADLR, the NH moiety of the pyrrole ring, independently of the position in the pyrazolobenzotriazine core, fits in HBp-3 area and points out the acceptor feature of this hydrogen bond area, already known as donor area. Unexpectedly, the oxygen atom of the furane ring does not form efficient hydrogen bond with the same area, probably for an imperfect distance. The size of substituent at position 8 is important but not necessary for the receptor recognition, in fact the interdependence between the features of the 3- and 8-substituent's is again verified, (i.e., compound 20 vs 32).

Combined Use of Cyclodextrins and Amino Acids for the Development of Cefixime Oral Solutions for Pediatric Use.

Cefixime (CEF) is a cephalosporin included in the WHO Model List of Essential Medicines for Children. Liquid formulations are considered the best choice for pediatric use, due to their great ease of administration and dose-adaptability. Owing to its very low aqueous solubility and poor stability, CEF is only available as a powder for oral suspensions, which can lead to reduced compliance by children, due to its unpleasant texture and taste, and possible non-homogeneous dosage. The aim of this work was to develop an oral pediatric CEF solution endowed with good palatability, exploiting the solubilizing and taste-masking properties of cyclodextrins (CDs), joined to the use of amino acids as an auxiliary third component. Solubility studies indicated sulfobutylether-ß-cyclodextrin (SBEßCD) and Histidine (His) as the most effective CD and amino acid, respectively, even though no synergistic effect on drug solubility improvement by their combined use was found. Molecular Dynamic and 1H-NMR studies provided insight into the interactions of binary CEF:His and ternary CEF:His:SBEßCD systems used to prepare CEF solutions, which resulted stable and maintained unchanged antimicrobial activity during the two-weeks-use in therapy. The ternary solution was superior in terms of more tolerable pH (5.6 vs. 4.7) and better palatability, being resulted completely odorless by a panel test.

Does Fermentation Really Increase the Phenolic Content in Cereals? A Study on Millet.

Millet is underutilized in Europe, despite its advantages compared to other common cereals. In Asia and Africa, millet is mainly eaten in fermented form; its consumption has beneficial properties on human health. Three millet batches were compared in terms of free and bound phenols by High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry (HPLC-DAD-MS). The richest one in terms of bound phenols was selected for testing via a basic (0.1 M NaOH) and an acidic (1.2 M H2SO4) hydrolysis, in which 149.3 and 193.6 mg/100 g of phenols were recovered, respectively. The ability of fermentation, with yeast and Lactobacilli, to increase the content of phenolic compounds was verified. Five withdrawalswere performed to verify the influence of fermentation time on the total phenolic content. The greatest phenolic content was observed after 72 h. Fermentation increased the cinnamic acids and flavonoids contents by approximately 30%. Vitexin and vitexin 2″-O-rhamnoside contents were significantly higher in the fermented millet; these compounds partially inhibit the protein tyrosine phosphatase enzyme, which is overexpressed in type-2 diabetes. A molecular dynamic simulation showed the two flavonoids to be allosteric inhibitors. The phenolic extract from fermented millet demonstrated a higher level of antioxidant protection on human erythrocytes by ex vivo cellular antioxidant activity in red blood cells. In this context, functional foods based on fermented millet could represent a new trend in European markets.

Headspace Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry Quantification of the Volatile Profile of More than 1200 Virgin Olive Oils for Supporting the Panel Test in Their Classification: Comparison of Different Chemometric Approaches.

A reliable and robust tool for supporting the panel test in virgin olive oil classification is still required. We propose four chemometric approaches based on t test, principal component analysis (PCA) and linear discriminant analysis (LDA), applied for combining sensorial data, and chemical measurements. The former was from the panel test, and the latter was from headspace solid-phase microextraction-gas chromatography-mass spectrometry quantitation of 73 volatile organic compounds (VOCs) of 1223 typical commercial virgin olive oils, with most of them recognized as difficult to classify with accuracy by the panel test. The approaches were developed and validated, and the best results, with 83.5% correct classification, were using the PCA-LDA approach. Among the other methods, developed for proposing simplified procedures based on a smaller number of VOCs, the best method gave 80.1% correct classification only using 10 VOCs. All of the approaches suggested that octane, heptanal, pent-1-en-3-ol, Z-3-hexenal, nonanal, and 4-ethylphenol should be considered as a basis of volatiles for classification of olive oil samples.

Synthesis, SAR and in vitro evaluation of new cyclic Arg-Gly-Asp pseudopentapeptides containing a s-cis peptide bond as integrin alphavbeta3 and alphavbeta5 ligands.

The solid-phase synthesis of two diastereomeric cyclic pseudopeptides containing the Arg-Gly-Asp sequence and the dipeptide isostere 2-amino-3-oxotetrahydro-1H-pyrrolizine-7a(5H)-carboxylic acid (GPTM) is described. Competition binding assays to purified alphavbeta3 and alphavbeta5 integrins with respect to [125I]echistatin showed a high inhibitory activity for the (2S,7aS)-GPTM derivative. Effects of the structural constraint induced by the two enantiomeric scaffolds (2R,7aR)-GPTM and (2S,7aS)-GPTM on the conformation of Arg-Gly-Asp sequence have been computationally investigated using as a reference the recently solved X-ray structure of cyclo(Arg-Gly-Asp-d-Phe-[N-Me]Val) in complex with the extracellular fragment of the alphavbeta3 receptor. The computational method disclosed the key role played by a bridging water molecule on differentiating the two ligands by a diverse stabilization of the ligand-protein complex.

Novel 3-aroylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted, ligands at GABAA/benzodiazepine receptor complex: synthesis, pharmacological and molecular modeling studies.

The synthesis and binding studies of a series of 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted are reported. High-affinity ligands at benzodiazepine site on GABA(A) receptor complex (GABA(A)/BzR complex) were obtained when the 3-aroyl substituent is represented by a five-member heteroaroyl ring (furoyl-, thenoyl-, and pyrroyl-). Moreover the type of heteroaroyl ring at position 3 influences the feature of the substituent at position 8 to obtain high-affinity ligands: a 'hydrogen-bond acceptor ring' at position 3 is synergic with an electron donor substituent at position 8, while a 'hydrogen-bond donor ring' is synergic with a withdrawing substituent. Compounds 8a, 9b, and 11 were deeply studied in vivo for their pharmacological effects considering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motor activity and explorative activity, anxiolytic-like effects, mouse learning and memory modulation, and ethanol-potentiating action. To rationalize and qualitatively interpret the GABA(A)/Bz binding affinities of compounds 8a and 11, a dynamic molecular modeling study has been performed, with the aim of assessing the preferred geometry of protein-ligand complex.

Exploring the intermolecular interactions acting in solvent-modified MEKC by Molecular Dynamics and NMR: The effect of n-butanol on the separation of diclofenac and its impurities.

An integrated approach involving CE experiments, Molecular Dynamics (MD) simulations and two-dimensional NOE spectroscopy (2D-NOESY) experiments was employed to elucidate the intermolecular interactions and the separation mechanisms involved in a solvent-modified MEKC method for the simultaneous determination of diclofenac sodium and its impurities. The CE findings indicated that the addition of n-butanol (nBuOH) to the SDS micellar solution played a primary role for controlling the partitioning into the mixed micelles and the migration of the analytes and that the presence of nBuOH as cosurfactant was compulsory for achieving the complete separation of the compounds. The different capacity factors of the analytes were calculated and a change in solute association with the mixed micelle when changing the SDS/nBuOH molar ratio was highlighted. The optimal SDS/nBuOH molar ratio for the electrophoretic separation was 1:8. On the other hand, both MD simulations and NMR experiments indicated that the most favorable molar ratio for the formation of mixed SDS/nBuOH micelles was 1:2. These results suggested that probably there is an excess of nBuOH in the background electrolyte, both as free molecules and in form of aggregates, which is able to interact with the analytes, and thus may compete with mixed micelles for the considered compounds. The calculated values of gain in potential energy of the analytes when included in mixed micelles were in agreement with the observed migration order of the compounds. The role of methyl-ß-cyclodextrin (MßCyD) in the background electrolyte was also investigated, since the addition of this CyD to the solvent-modified MEKC system was found to be useful to reduce the analysis time. MD simulations and 2D-NOESY spectra highlighted the formation of inclusion complexes with MßCyD not only with the analytes, but also with SDS. MßCyD may lower the availability of both SDS and nBuOH for forming micelles and mostly may compete with the mixed micelle as a second pseudostationary phase.