Comparison of Determination of Drug–Muscarinic Receptor Affinity by Cell-Membrane Chromatography and by Radioligand-Binding Assay with the Cerebrum Membrane of the Rat

This study was performed to determine whether cell-membrane chromatography (CMC) can be used to reflect the selectivity and specificity of interactions between drugs and the muscarinic acetylcholine receptor (mAChR). A cell membrane stationary phase (CMSP) was prepared by immobilizing rat cerebrum cell membrane on the surface of a silica carrier and used for rapid on-line chromatographic evaluation of ligand binding affinity to mAChR. Comparison of the results with affinity rank orders obtained from radioligand-binding assays using the same cerebrum membrane indicated a positive correlation (r²=0.8640, n=18, P < 0.0001) between the data sets and proved that CMC can be used to evaluate drug–receptor affinity for drug candidates.Revised: 19 July and 15 November 2004     

Prostate Cell Membrane Chromatography�CLC/MS Method for Screening ��1A-Adrenoceptor Antagonists from Lotus Plumule

The ??_1A-adrenoceptor (??_1A-AR) plays an important role in drug discovery and development. An online analytical method coupling prostate cell membrane chromatography (CMC) with high-performance liquid chromatography/mass spectrometry (LC/MS) was established to screen ??_1A-AR antagonists in traditional Chinese medicines (TCMs). The prostate cell membrane stationary phase (CMSP) was prepared by immobilizing the prostate cell membrane onto the surface of the silica carrier. The surface and chromatographic characteristics of the prostate CMSP were studied using tamsulosin as a model molecule. The retained fractions from the prostate CMC were analyzed by transferring them to an LC/MS system through a 10-port switch valve. The active component, which could act on the prostate cell membrane and receptor on it (such as ??_1A-AR), was determined using a displacement experiment. The results indicated that liensinine, isoliensine, and neferine from Lotus Plumule exhibited similar retention characteristics to the control drug tamsulosin when utilizing the prostate CMC model. This new prostate CMC?CLC/MS method is applicable for screening ??_1A-AR antagonists from TCMs such as Lotus Plumule and could be employed as a drug discovery tool for natural medicinal herbs.     

A new A431/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening epidermal growth factor receptor antagonists from Radix sophorae flavescentis

The intracellular kinase domains of epidermal growth factor receptor (EGFR) in some tumor cells such as human epidermal squamous cells (A₄₃₁ cells) are an important target for drug discovery. We have developed a new A₄₃₁/cell membrane chromatography (A₄₃₁/CMC)-online–high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening EGFR antagonists from medicinal herbs such as traditional Chinese medicines (TCMs). In this study, A₄₃₁ cells with high EGFR expression levels were used to prepare cell membrane stationary phase (CMSP) in an A₄₃₁/CMC model. The retention fractions eluted from the CMSP column were enriched onto an ODS pre-column and then switched into an HPLC/MS system by combining a 10 port columns switching valve. The screening results found that oxymatrine and matrine from Radix sophorae flavescentis (RSF) were the targeted components which could act on EGFR in similar manner of gefitinib as a control drug. There was a good relationship of their inhibiting effects on EGFR secretion and A₄₃₁ cell growth in vitro. This new A₄₃₁/CMC-online-HPLC/MS method can be applied for screening EGFR antagonists from TCMs such as RSF. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

Establishment of the Model CD40 Cell Membrane Chromatography and Its Chromatographic Characteristics

A new model of atherosclerosis cell membrane chromatography has been established by using a CD40 cell membrane stationary phase (CD40 CMSP) prepared by immobilizing the CD40 cell membrane onto the surface of a silica carrier. The surface and chromatographic characteristics of CD40 CMSP were studied. The retention characteristics of anti-CD40 antibody and statins (lovastatin, simvastatin and pravastatin) were also investigated using this model. Affinities of the anti-CD40 antibody and statins toward CD40 cell membrane and receptors were based on the determination of log k′ values (the logarithm of capacity factor of a solute). There was a significant correlation between the affinity in the CD40–CMC and the effect in vitro for the pharmacological effect.     

Cell membrane chromatography competitive binding analysis for characterization of α<Subscript>1A</Subscript> adrenoreceptor binding interactions

A new high α_1A adrenoreceptor (α_1AAR) expression cell membrane chromatography (CMC) method was developed for characterization of α_1AAR binding interactions. HEK293 α_1A cell line, which expresses stably high levels of α_1AAR, was used to prepare the stationary phase in the CMC model. The HEK293 α_1A/CMC-offline-HPLC system was applied to specifically recognize the ligands which interact with the α_1AAR, and the dissociation equilibrium constants (K _D) obtained from the model were (1.87 ± 0.13) × 10⁻⁶ M for tamsulosin, (2.86 ± 0.20) × 10⁻⁶ M for 5-methylurapidil, (3.01 ± 0.19) × 10⁻⁶ M for doxazosin, (3.44 ± 0.19) × 10⁻⁶ M for terazosin, (3.50 ± 0.21) × 10⁻⁶ M for alfuzosin, and (7.57 ± 0.31) × 10⁻⁶ M for phentolamine, respectively. The competitive binding study between tamsulosin and terazosin indicated that the two drugs interacted at the common binding site of α_1AAR. However, that was not the case between tamsulosin and oxymetazoline. The results had a positive correlation with those from radioligand binding assay and indicated that the CMC method combined modified competitive binding could be a quick and efficient way for characterizing the drug–receptor interactions.     

Synthesis of 6α-methyl-16α,17α-cyclohexano-19-norprogesterone from a 19-methyl-6-desmethyl precursor

After prolonged refluxing of 19-tosyloxy-16α,17α-cyclohexanopregn-5-en-3β-ol-20-one (3) with NaI in 2-propanol, the initially formed 19-iodo derivative (4) undergoes supraface migration of the CH₂I group from the C(10) atom to the C(6) atom, probably through involvement of a homoallyl cation. The resulting 6β-iodomethyl-16α,17α-cyclohexano-19-norpregn-5(10)-en-3β-ol (5) was transformed in three steps into 6α-methyl-16α,17α-cyclohexano-19-norprogesterone (6α-methyl-19-nor-D′ ₆-pentarane,8). The transformation of compound5 into the target product8 also gave a side product, a pentarane with aromatic ringA (10), which was isolated and characterized by spectroscopic methods. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1688–1691, September, 1997.     

An electroconductivity study on degree of counterion binding or dissociation of a-sulfonatomyristic acid methyl ester micelles in water as a function of temperature

 For a sodium salt of α-sulfonatomyristic acid methyl ester (14SFNa), one of the α-SFMe series surfactants, the differential conductivity (∂κ/∂C) _T , _P vs. square root of concentration (√C) was employed in order to determine not only CMC but also the limiting molar conductance (Λ⁰) and the molar conductance of micellar species (Λ^M). Based on the data of the degree of counterion binding to micelles (β) determined previously at different temperatures ranging 15–50 °C at every 5 °C, the experimental values of the degree of dissociation (ionization) of a micelle (α_EX) were calculated by regarding as α_EX=1−β. The ratio Λ^M/Λ⁰ corresponding to the ratio of slopes below and above CMC in the curve of specific conductivity (κ) vs. concentration (C), which has been often assumed to be the degree of ionization of micelles (α), was compared with the present α_EX. However, the ratio Λ^M/Λ⁰ (=α) was found to have a correlationship with α_EX (=1−β) as α_EX≈0.40×(Λ^M/Λ⁰), or strictly, α_EX=0.40 (Λ^M/Λ⁰)+0.08, indicating that the simple ratio of the slopes below and above CMC in κ vs. C curve is not true for α_EX=1−β. On the other hand, the method proposed by Evans gave a value closer to α_EX compared with the simple ratio. Received: 17 September 1996 Accepted: 8 April 1997     

Hydrogels from diacylphosphatidylcholine

The formation of hydrogels from diacylphosphatidylcholine (PC) and water/glycerol mixtures and the properties of the gels are reported. The gels are formed when L_α phases from the PC in the solvent mixtures are cooled from T >55 °C below the Krafft temperature of the PC (T _m ∼52 °C). The glycerol can also be replaced by other co-solvents like butylenglycol. Above T _m, the PC spontaneously forms L_α phases with multilamellar vesicles that show a strong stationary birefringence. On cooling below T_m, the L_α phases jellify to transparent gels. DSC measurements of the gels show that the PC molecules undergo a phase transition into the crystalline state. This transition does not seem to be accompanied by a change of the morphological structure of the liquid L_α phase. The hydrogels also have a stationary birefringence. The vesicles in the gels have been imaged by the CryoTEM method. The hydrogels are already formed with as little as 1% of PC in the mixed solvent. The rheological properties of the gels were determined from oscillating rheological measurements. Samples with 10% of PC have a storage modulus of >10,000 Pa.     

Stereochemical effects during [M-H]− dissociations of epimeric 11-OH-17β-estradiols and distant electronic effects of substituents at C(11) position on gas phase acidity

The affinity of estradiol derivatives for the estrogen receptor (ER) depends strongly on nature and stereochemistry of substituents in C₍₁₁₎ position of the 17β-estradiol (I). In this work, the stereochemistry effects of the 11α-OH-17β-estradiol (III_α) and 11β-OH-17β-estradiol (III_β) were investigated using CID experiments and gas-phase acidity (ΔH_acid^∘) determination. The CID experiments showed that the steroids decompose via different pathways involving competitive dissociations with rate constants depending upon the α/β C₍₁₁₎ stereochemistry. It was shown that the fragmentations of both deprotonated [III_α-H]⁻ and [III_β-H]⁻ epimers were initiated by the deprotonation of the most acidic site, i.e. the phenolic hydroxyl at C₍₃₎. This view was confirmed by H/D exchange and double resonance experiments. Furthermore, the ΔH_acid^∘ of both epimers (III_α and III_β), 17β-estradiol (I), and 17-desoxyestradiol (II) was determined using the extended Cooks’ kinetic method. The resulting values allowed us to classify steroids as a function of their gas-phase acidity as follows: (III_β)≫(II)>(I)>(III_α). Interestingly, the α/β C₍₁₁₎ stereochemistry appeared to influence strongly the gas-phase acidity. This phenomenon could be explained through stereospecific proton interaction with π-orbital cloud of A ring, which was confirmed by theoretical calculation.     

Investigation of calcium antagonist–L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method

The dissociation equilibrium constant (K _D) is an important affinity parameter for studying drug–receptor interactions. A vascular smooth muscle (VSM) cell membrane chromatography (CMC) method was developed for determination of the K _D values for calcium antagonist–L-type calcium channel (L-CC) interactions. VSM cells, by means of primary culture with rat thoracic aortas, were used for preparation of the cell membrane stationary phase in the VSM/CMC model. All measurements were performed with spectrophotometric detection (237 nm) at 37 °C. The K _D values obtained using frontal analysis were 3.36 × 10⁻⁶ M for nifedipine, 1.34 × 10⁻⁶ M for nimodipine, 6.83 × 10⁻⁷ M for nitrendipine, 1.23 × 10⁻⁷ M for nicardipine, 1.09 × 10⁻⁷ M for amlodipine, and 8.51 × 10⁻⁸ M for verapamil. This affinity rank order obtained from the VSM/CMC method had a strong positive correlation with that obtained from radioligand binding assay. The location of the binding region was examined by displacement experiments using nitrendipine as a mobile-phase additive. It was found that verapamil occupied a class of binding sites on L-CCs different from those occupied by nitrendipine. In addition, nicardipine, amlodipine, and nitrendipine had direct competition at a single common binding site. The studies showed that CMC can be applied to the investigation of drug–receptor interactions.     

Cell Membrane Chromatography Correlated with Functional Assay for Ligand–β-Adrenergic Receptor Affinities

This study was performed to investigate whether the retention factor (k) from cell membrane chromatography (CMC) can be used to assess the affinity of ligands to β-adrenergic receptor (β-AR) and the correlationship between the factor and pharmacoligical effects. The cell membrane of guinea pig myocardium membrane was immobilized on the surface of the silica carrier as the cell membrane stationary phase (CMSP) for the rapid on-line chromatographic evaluation of ligand binding affinity to β-AR. The affinity was also evaluated by functional assay using the same tissues. Correlation analysis was used to assess the correlationship of these two methods. The retention factors in guinea pig myocardium CMSP were: (−)-propranolol (33.9) > (+)-propranolol (27.0) > metopranolol (23.2) > esmolol (17.7) > practolol (13.2) > sotalol (9.56). Compared to the affinity rank orders obtained from functional assay in the same myocardium, there was a positive correlation (r ² = 0.9729, n = 18, p < 0.0001) between both data sets. These results showed that CMC can be used to evaluate drug–receptor affinities of drug candidates as the functional assays.

     

On-line concentration by field-enhanced sample injection with reverse migrating micelles in micellar electrokinetic capillary chromatography for the analysis of triterpenoids from traditional chinese medicine

In this work, a simple, reproducible and sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of five triterpenoids, lupeol (1), 1β-hydroxy-lupeol (2), lup-3β,1α-diol (3), lup-1β,3β,11α-triol (4) and 30-norlupan-3β,11α-diol-20-one (5) in traditional Chinese medicine of Salvia roborowskii Maxim. Field-enhanced sample injection with reverse migrating micelles (FESI-RMM) was used for on-line concentration of triterpenoids. The optimum buffer contained 50 mM H₃PO₄, 160 mM SDS, 20% acetonitrile and 15% 2-propanol and pH of buffer was 2.0. The sample solution was diluted with 10 mM H₃PO₄ (pH 2.5, containing 10 mM SDS) and injected for 15 s with −8 kV after injection of 4 s water plug. The effects of concentrations of sodium dodecyl sulfate (SDS) and organic modifier, the sample matrix, the injection time of water plug, the injection voltage and injection time of sample on the separation and stacking efficiency were investigated. Under the optimum conditions, the analytes were well separated and by optimizing the stacking conditions, about 28–96-fold improvement in the detection sensitivity was obtained for triterpenoids. The contents of five triterpenoids in Salvia roborowskii Maxim were successfully determined with satisfactory repeatability and recovery.     

Cell Membrane Chromatography Correlated with Functional Assay for Ligand–β-Adrenergic Receptor Affinities

This study was performed to investigate whether the retention factor (k) from cell membrane chromatography (CMC) can be used to assess the affinity of ligands to β-adrenergic receptor (β-AR) and the correlationship between the factor and pharmacoligical effects. The cell membrane of guinea pig myocardium membrane was immobilized on the surface of the silica carrier as the cell membrane stationary phase (CMSP) for the rapid on-line chromatographic evaluation of ligand binding affinity to β-AR. The affinity was also evaluated by functional assay using the same tissues. Correlation analysis was used to assess the correlationship of these two methods. The retention factors in guinea pig myocardium CMSP were: (?)-propranolol (33.9) > (+)-propranolol (27.0) > metopranolol (23.2) > esmolol (17.7) > practolol (13.2) > sotalol (9.56). Compared to the affinity rank orders obtained from functional assay in the same myocardium, there was a positive correlation (r ² = 0.9729, n = 18, p < 0.0001) between both data sets. These results showed that CMC can be used to evaluate drug–receptor affinities of drug candidates as the functional assays.     

Structural and spectroscopic characterization of Al2−x Cr x O3 powders obtained by polymeric precursor method

Al₂O₃ and Al_2−x Cr _x O₃ (x = 0.01, 0.02 and 0.04) powders have been synthesized by the polymeric precursors method. A study of the structural evolution of crystalline phases corresponding to the obtained powders was accomplished through X-Ray Diffraction and UV-vis spectroscopy (reflectance spectra and CIEL^*a^*b^* color data). The obtained results allow to identify the γ-Al₂O₃ to α-Al₂O₃ phase transition. The single-phase α-Al₂O₃ powder was obtained after heat treatment at 1050 °C for 2 h. The results show that the green to red color transition and ruby luminescence lines observed for the powders of Al_2−x Cr _x O₃ are related to the γ to α-Al₂O₃ phase transition and the temperature and time range for such transition depends on the chromium content.     

Interactions between anionic mixed micelles and α-cyclodextrin and their inclusion complexes: conductivity, NMR and fluorescence study

Mixed micelle formation of anionic surfactants sodium dodecyl sulfate (SDS) and sodium lauroyl sarcosine (SLAS) have been studied in water and in 5, 10, and 15 mM concentrations of α-cyclodextrin (α-CD) over mole fraction range of α _SDS from 0 to 1. From the conductivity curves, the critical micellar concentration (CMC) for the pure and binary mixtures were evaluated. The degree of counterion association (χ) or counterion dissociation (δ), the equivalent ionic conductivities of the monomeric species (Λ _m), the associated species (Λ _assc), and the micelle (Λ _mic) were evaluated from the slope of the conductivity vs concentration plots. The CMC values have been used to calculate the thermodynamic parameters such as the standard free energy of micelle formation and a transfer of standard free energy of micelle from the aqueous medium to additive medium computed. The apparent CMC of the surfactants varies linearly with α-CD concentrations. From the dependence of CMC of the surfactants on α-CD concentration, we are able to determine the association constant (K) of surfactant-α-CD inclusion complexes assuming 1:1 stoichiometry. Mixed micelle behaves ideally in the pure water as well as at the different concentrations of α-CD, which was evaluated by using the Clint equation, the regular solution approximation, and Motomura’s formulation. Self-diffusion coefficients of the micelle increased upon the induction of SDS into the micelle. 2D-rotating frame Overhauser effect spectroscopy spectra of SDS and SLAS were recorded in the presence of α-CD to investigate the interaction between H-atoms of the alkyl chain of the surfactants and H-atoms of the hydrophobic cavity of α-CD indicating multiple complexation. The fluorescence anisotropy of rhodamine B has been measured to observe the structural behavior of mixed micelle.     

Phase transition in LAMOX type compounds

La₂Mo₂O₉ (LMO) was synthesized at lower temperature 973 K (LT-phase) by ceramic route. Differential thermal analysis (DTA) scan of LT-phase of LMO showed α→β transition at 843 K during heating and β→α conversion via a metastable γ-phase during cooling. This was also confirmed by thermo-dilatometry and impedance spectroscopy. La₂Mo_1.95V_0.05O_9-δ (LMVO), La_1.96Sr_0.04Mo₂O_9-δ (LSMO) and La_1.96Sr_0.04Mo_1.95V_0.05O_9-δ (LSMVO) were prepared in a similar way. These compounds exhibited α→β transition on heating with shift in transition temperature, but the existence of γ-phase during cooling disappeared. Substitution increased the ionic conductivity of α-phase and reduced that of β-phase.     

Employing perichromism for probing the properties of carboxymethyl cellulose films: an expedient, accurate method for the determination of the degree of substitution of the biopolymer derivative

The properties of films of carboxymethyl cellulose, CMC, of different degree of substitution, DS, have been examined by the use of perichromic indicators (probes). The film properties that have been determined are: empirical polarity, E_T(33); “acidity”, α; “basicity”, β; and dipolarity/polarizability, π*. This has been achieved by employing the following perichromic probes: 4-nitroaniline, 4-nitroanisole, 4-nitro-N,N-dimethylaniline, and 2,6-dichloro-4-(2,4,6-triphenyl-pyridinium-1-yl)phenolate, WB. The correlations between both E_T(33)- or π* and DS were found to be linear; that between β and DS is a second order polynomial; no obvious correlation was found between α and DS. The polarities of CMC films are in the range of those of butyl alcohols. As models for CMC, we have employed cellulose plus CMC of high DS; oxidized cellulose with degree of oxidation = 0.5; sodium glucuronate. The former model behaved akin to CMC, but the plots of the perichromic properties versus DS showed different slopes/intercepts. FTIR data and molecular dynamics simulations on the solvation of WB have shown that this difference can be traced to more efficient hydrogen bonding between the film of the model and the probe. This affects the intra-molecular charge-transfer energy of the latter, leading to different responses to the variation of DS. Based on the excellent linear correlation between E_T(33) and DS, for CMC from different origins, we suggest that perichromism is a simple, accurate, and expedient alternative for the determination of DS of the biopolymer derivative.     

<Emphasis Type="Italic">iso</Emphasis>-<Emphasis Type="Italic">α</Emphasis>-Cyclopiazonic acid,a new natural product isolated from the marine-derived fungus <Emphasis Type="Italic">Aspergillus flavus</Emphasis> C-F-3

A new natural product, iso-α-cyclopiazonic acid (1), together with its isomer α-cyclopiazonic acid (2); three mycotoxins: aflatoxin B₁ (AFB₁) (3), aflatoxin Q₁ (AFQ₁) (4), and O-methylsterigmatocystin (OMST) (5); two diketopiperazine alkaloids: ditryptophenaline (6) and 3-[(1H-indol-3-yl)methyl]-6-benzylpiperazine-2,5-dione (7), were isolated from the marine-derived fungus Aspergillus flavus. Their structures were determined by analysis of spectroscopic data. The cytotoxicities of compounds 1 and 2 were studied using HL-60, MOLT-4, A-549, and BEL-7402 cell lines.     

Preparation,characterization and thermal decomposition of phenylenediammonium sulfate salts Part. XVI

Polyaniline/α-Al₂O₃ (PANI/α-Al₂O₃) composites were synthesized by in situ polymerization through ammonium persulfate ((NH₄)₂S₂O₈, APS) oxidized aniline using HCl as dopant. XRD and FTIR were used to characterize the PANI/α-Al₂O₃ composites. The thermal stabilities and glass transition temperature (T _g) of PANI/α-Al₂O₃ composites were tested using thermogravimetric (TG) method and modulated differential scanning calorimetry (MDSC) technique. The results of TG showed that the thermal stability of PANI/α-Al₂O₃ composite increased and then decreased with the increase in α-Al₂O₃ content. The derivative thermogravimetry (DTG) curves showed one step degradation of PANI when the α-Al₂O₃ content was lower than 52.5 mass%, and exhibited two steps degradation when the α-Al₂O₃ content was higher than 63.6 mass%. The MDSC curves showed that the T _g of PANI/α-Al₂O₃ composites increased and then decreased with the augment of α-Al₂O₃ for the interaction between PANI chains and the surface of α-Al₂O₃.     

Kinetic Analysis of Thermal Decomposition of Ca(OH)2 Formed During Hydration of Commercial Portland Cement by DSC

In this paper, evaluation of kinetic parameters (the activation energy – E,the pre-exponential factor – A and the reaction order – n) with simultaneous determination of the possible reaction mechanism of thermal decomposition of calcium hydroxide (portlandite), Ca(OH)₂ formed during hydration of commercial Portland-slag cement, by means of differential scanning calorimetry (DSC) in non-isothermal conditions with a single heating–rate plot has been studied and discussed. The kinetic parameters and a mechanism function were calculated by fitting the experimental data to the integral, differential and rate equation methods. To determine the most probable mechanism, 30 forms of the solid-state mechanism functions, f(α_c) have been tried. Having used the procedure developed and the appropriate program support, it has been established that the non-isothermal thermal decomposition of calcium hydroxide in the acceleratory period (0.004<α_c<0.554) can be described by the rate equation: d α_c/dT=A/βexp(−E/RT)f(α_c), which is based on the concept of the mechanism reaction:f(α_c)=2(α_c)^1/2. The mechanism functions as well as the values of the kinetic parameters are in good agreement with those given in literature. This revised version was published online in August 2006 with corrections to the Cover Date.