Comparing the predictability of different chemometric models over UV-spectral data of isoxsuprine and its toxic photothermal degradation products.

Isoxsuprine (ISX) is widely used for cerebral and peripheral vascular diseases. A comparative study was held among different multivariate calibration models for selective determination of a complex mixture of Isoxsuprine and four of its toxic photothermal degradation products that impair kidney and liver functions. The Partial Least Squares (PLS) and Artificial Neural Network (ANN) models were applied on the specific spectrum and on selected wavelengths using genetic algorithm (GA) technique as an efficient variable selection tool. The effect of GA on the model construction and performance was evaluated. The multilevel multifactor experimental design was adopted for the construction of the calibration set. Optimized parameters were used for the development of the different models. The performances of the developed models were assessed by predicting the concentration of eight different mixtures composing the validation set. Results were compared to one another and to the official method using one-way ANOVA statistical test to assure the validity of the constructed models. The lower chance of overfitting offered by ANN minimized the RMSEP relative to the PLS. On the other hand, the application of GA prior to model implementation affected the number of latent variables the prediction ability of both PLS and ANN models. The validated models were successfully applied as stability indicating assay methods for the selective determination of ISX and its photothermal degradation products in ISX raw material and market formulations.

Vasodilation Elicited by Isoxsuprine, Identified by High-Throughput Virtual Screening of Compound Libraries, Involves Activation of the NO/cGMP and H2S/KATP Pathways and Blockade of α1-Adrenoceptors and Calcium Channels.

Recently, our research group demonstrated that uvaol and ursolic acid increase NO and H2S production in aortic tissue. Molecular docking studies showed that both compounds bind with high affinity to endothelial NO synthase (eNOS) and cystathionine gamma-lyase (CSE). The aim of this study was to identify hits with high binding affinity for the triterpene binding-allosteric sites of eNOS and CSE and to evaluate their vasodilator effect. Additionally, the mechanism of action of the most potent compound was explored. A high-throughput virtual screening (HTVS) of 107,373 compounds, obtained from four ZINC database libraries, was performed employing the crystallographic structures of eNOS and CSE. Among the nine top-scoring ligands, isoxsuprine showed the most potent vasodilator effect. Pharmacological evaluation, employing the rat aorta model, indicated that the vasodilation produced by this compound involved activation of the NO/cGMP and H2S/KATP signaling pathways and blockade of α1-adrenoceptors and L-type voltage-dependent Ca2+ channels. Incubation of aorta homogenates in the presence of isoxsuprine caused 2-fold greater levels of H2S, which supported our preliminary in silico data. This study provides evidence to propose that the vasodilator effect of isoxsuprine involves various mechanisms, which highlights its potential to treat a wide variety of cardiovascular diseases.

Binding interaction of isoxsuprine hydrochloride and levothyroxine to milk ß-lactoglobulin; from the perspective of comparison.

Isoxsuprine hydrochloride (ISO) and levothyroxine (LEV) are medicines which can be utilized alone or simultaneously by pregnant women. The purpose of this work is to investigate the separate and simultaneous interaction of ISO and LEV with ß-LG. The results showed that both drugs can bind to ß-LG; the static quenching was suggested for fluorescence quenching mechanism of ß-LG.The values of binding constants (Kß-LG-ISO = 2.69 × 104 M-1, Kß-LG-LEV = 0.54 × 103 M-1 and Kß-LG-ISO-LEV = 2.18 × 103 M-1 at 310 K) suggested that ISO has stronger binding affinity toward ß-LG than LEV and affinity of ß-LG to LEV is increased in the presence of ISO while the presence of LEV has no significant effect on the affinity of protein to ISO. Thermodynamic parameters showed that the binding of LEV to ß-LG are hydrogen bonding and Van der Waals forces but the formation of ß-LG-ISO is hydrophobic associations. The results of FT-IR and UV-visible measurements indicated that the binding of both drugs to ß-LG may induce conformational changes of protein. In silico molecular docking analyses confirmed that ISO and LEV binds to residues located at site I and site II of ß-LG, respectively.

Binding Studies of Isoxsuprine Hydrochloride to Calf Thymus DNA Using Multispectroscopic and Molecular Docking Techniques.

In the present work, the interaction of Isoxsuprine (ISX) with Calf thymus DNA (ct-DNA) under physiological conditions (Tris-HCl buffer of pH 7.4) was investigated by using electronic absorption, circular dichroism, viscosity, electrochemical studies, fluorescence techniques, salt effect studies and computational studies. Competitive fluorimetric studies with Hoechst 33258 have shown that ISX exhibit the ability to displace the DNA-bound Hoechst 33258, indicating that it binds to ct-DNA in strong competition with Hoechst 33258 for the minor groove binding. Furthermore, the resulting data showed that ISX cannot displace methylene blue or acridine orange, which are the common intercalator molecules. The viscosity of ct-DNA solution was almost unchanged on addition of ISX and circular dichroism (CD) spectra of ct-DNA showed small changes in the presence of ISX which is in agreement with groove binding mode of interaction. Thus all above studies showed that the ISX drug binds to ct-DNA in a groove binding mode.The salt-effect studies showed the non-electrostatic nature of binding of ISX to ct-DNA. Moreover, molecular docking results support the above experimental data and suggest that ISX prefers to bind on the minor groove of ct-DNA.

Comparative Study Between Zero-Order Spectra-Processing and Ratio Spectra-Manipulating Methods Applied for the Determination of Isoxsuprine Hydrochloride in the Presence of Its Oxidative Degradation Product.

Four accurate, precise, and validated stability-indicating spectrophotometric methods handling either zero-order spectra or ratio spectra have been developed and compared for the analysis of isoxsuprine hydrochloride (ISX) in the presence of its oxidative degradation product. The first two methods processed zero-order spectra, namely graphical absorbance ratio or Q-Analysis and area under the curve, whereas the third and fourth methods manipulated ratio spectra, namely the ratio difference spectrophotometric method and derivative ratio. The proposed methods showed good linearity in the range of 2-23 µg/mL. The methods were tested for specificity using laboratory-prepared mixtures containing the drug and its degradation product. The proposed methods were applied for the determination of ISX in Vascular tablets and the obtained results were acceptable, with small percentage RSD values. The validity of the proposed procedures was further assessed by applying the standard addition technique, which showed no interference from excipients. The obtained results were statistically compared with those obtained by the reported method, showing no significant differences when t- and F-tests were applied.

Exploring isoxsuprine hydrochloride binding with human serum albumin in the presence of folic acid and ascorbic acid using multispectroscopic and molecular modeling methods.

Isoxsuprine hydrochloride (vasodilator drug), folic acid and ascorbic acid are medicines which can be utilized alone or simultaneously by pregnant women. In the present work the competitive binding of isoxsuprine hydrochloride (ISO) with human serum albumin (HSA) in the absence and presence of folic acid (FOL) and ascorbic acid (AS) was investigated using different spectroscopic probes and molecular docking studies. The results of fluorescence suggested that isoxsuprine alone or in the presence of ascorbic acid can bind to HSA and quench the fluorescence of HSA with static mechanism but For HSA-folic acid-isoxsuprine system, dynamic type of quenching mechanisms is involved. The values of binding constants (KHSA-ISO~1.2×103M-1, KHSA-AS-ISO~2.1×103M-1and KHSA-FOL-ISO~0.7×103M-1) suggested that affinity of HSA to isoxsuprine increased in the presence of ascorbic acid while the presence of folic acid reduced the affinity of protein to isoxsuprine. The results of FT-IR and circular dichroism measurements indicated that the binding of isoxsuprine to HSA in the absence and the presence of folic acid and ascorbic acid may induce conformational and microenvironmental changes of protein. Not only do these types of spectroscopy techniques provide all the information about the systems, molecular docking, also emphasizes the results and is employed for the identification of the active site residues, bioactive conformer of Isoxsuprine and their critical interactions.

Spectrofluorimetric determination of certain adrenergic agonist drugs in their pure forms and pharmaceutical formulations: Content uniformity test application.

A new, simple, sensitive and rapid spectrofluorimetric method has been developed for determination of certain adrenergic agonists such as isoxsuprine hydrochloride, ritodrine hydrochloride and etilefrine hydrochloride in their pure forms and pharmaceutical dosage forms. The method depends on micellar enhancement of the native fluorescence of investigated drugs by using 2% w/v sodium dodecyl sulfate (SDS) as an anionic surfactant. The enhanced fluorescence intensity of investigated drugs was measured at 305 nm after excitation at 278 nm. The interaction of studied drugs with SDS was studied, and the enhanced fluorescence intensity was exploited to develop an assay method for the determination of investigated drugs. The relative fluorescence intensity-concentration plots were rectilinear over the range 0.15-3.00 µg ml-1 , with low quantification limits of 0.132, 0.123 and 0.118 µg mL-1 for isoxsuprine, ritodrine and etilefrine, respectively. The proposed method was successfully applied for determination of studied drugs in their pharmaceutical formulations. Moreover, the high sensitivity of the proposed method allows performing the content uniformity testing of the studied drugs in their tablets by using the official United States Pharmacopeia (USP) guidelines. Statistical comparisons of the results with those of the reported methods revealed excellent agreement and indicated no significant difference in accuracy and precision.

35Cl dynamic nuclear polarization solid-state NMR of active pharmaceutical ingredients.

In this work, we show how to obtain efficient dynamic nuclear polarization (DNP) enhanced 35Cl solid-state NMR (SSNMR) spectra at 9.4 T and demonstrate how they can be used to characterize the molecular-level structure of hydrochloride salts of active pharmaceutical ingredients (APIs) in both bulk and low wt% API dosage forms. 35Cl SSNMR central-transition powder patterns of chloride ions are typically tens to hundreds of kHz in breadth, and most cannot be excited uniformly with high-power rectangular pulses or acquired under conditions of magic-angle spinning (MAS). Herein, we demonstrate the combination of DNP and 1H-35Cl broadband adiabatic inversion cross polarization (BRAIN-CP) experiments for the acquisition of high quality wideline spectra of APIs under static sample conditions, and obtain signals up to 50 times greater than in spectra acquired without the use of DNP at 100 K. We report a new protocol, called spinning-on spinning-off (SOSO) acquisition, where MAS is applied during part of the polarization delay to increase the DNP enhancements and then the MAS rotation is stopped so that a wideline 35Cl NMR powder pattern free from the effects of spinning sidebands can be acquired under static conditions. This method provides an additional two-fold signal enhancement compared to DNP-enhanced SSNMR spectra acquired under purely static conditions. DNP-enhanced 35Cl experiments are used to characterize APIs in bulk and dosage forms with Cl contents as low as 0.45 wt%. These results are compared to DNP-enhanced 1H-13C CP/MAS spectra of APIs in dosage forms, which are often hindered by interfering signals arising from the binders, fillers and other excipient materials.

Resolution and isolation of enantiomers of (±)-isoxsuprine using thin silica gel layers impregnated with L-glutamic acid, comparison of separation of its diastereomers prepared with chiral derivatizing reagents having L-amino acids as chiral auxiliaries.

Thin silica gel layers impregnated with optically pure l-glutamic acid were used for direct resolution of enantiomers of (±)-isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l-alanine, l-valine and S-benzyl-l-cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin-layer chromatography (TLC) on reversed phase (RP) C18 plates. Diastereomers prepared with enantiomerically pure (+)-isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)-isoxsuprine. The elution order in the experimental study of RP-TLC and RP-HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1-0.09 µg/mL in TLC while it was in the range of 22-23 pg/mL in HPLC and 11-13 ng/mL in RP-TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification.

Investigation of ractopamine molecularly imprinted stir bar sorptive extraction and its application for trace analysis of beta2-agonists in complex samples.

In this paper, a novel molecularly imprinted polymer (MIP) coated stir bar with ractopamine as template by glass capillary filling with magnetic core as substrate was prepared reproducibly. The ractopamine MIP coating was homogeneous and porous with the average thickness of 20.6 microm. The extraction apparatus for the stir bar was improved to avoid coating loss. The MIP-coated stir bar showed better extraction capacity and good selectivity than that of non-imprinted polymer (NIP) coated stir bar to ractopamine and its analogues. The extraction capacities of ractopamine, isoxsuprine, clenbuterol and fenoterol for MIP-coated stir bar were 3.3, 3.1, 2.8 and 2.4 times as much as that of the NIP coated stir bar, respectively. The MIP-coated stir bars could be used at least 40 times without apparent damage and kept in dried air for 8 months without reduce of extraction ability. A method for the determination of beta(2)-agonists in complex samples by MIP-coated stir bar sorptive extraction coupled with high-performance liquid chromatography (HPLC) was developed. The linear ranges were 0.5-40 microg/L for ractopamine and 1.0-40 microg/L for isoxsuprine and clenbuterol. The detection limits were within the range of 0.10-0.21 microg/L. The method was successfully applied to the analysis of beta(2)-agonists in spiked pork, liver and feed samples with the recoveries of 83.7-92.3%, 80.5-90.2% and 73.6-86.2%, respectively. The RSDs was within 2.9-8.1%. The method is very suitable for the determination of trace beta(2)-agonists in pork, liver and feed samples.

Simultaneous determination of ritodrine and isoxsuprine using coupling technique of synchronous fluorimetry and H-point standard addition method.

Simultaneous determination of two structurally related ss(2) adrenergic receptor agonists namely, Ritodrine HCl (RTH) and Isoxsuprine HCl (ISP) was performed using coupling technique of synchronous fluorimetry and H-point standard addition method. Under optimum conditions, linear determination ranges were 1.48 - 14.80 x 10(-6) mol L(-1) and 1.54 - 15.44 x 10(-6) mol L(-1) for ISP and RTH respectively. RTH and ISP could be determined simultaneously without interference from each other when their concentration ratio varies from 5:1 to 1:5 in the mixed sample. The proposed method was applied to the determination of RTH and ISP in synthetic mixture of pharmaceutical samples, the accuracy and precision of the results were satisfactory.

Molecularly imprinted polymer for the determination of trace ractopamine in pork using SPE followed by HPLC with fluorescence detection.

In this study, a molecularly imprinted functionalized polymer for the selective separation of ractopamine (RAC) was prepared by combining a surface molecular imprinting technique with a sol-gel method process. The polymer was evaluated by static, kinetic adsorption, and selective experiments. Results indicated that the molecularly imprinted polymer had high adsorption capacity, selective ability, and fast mass transfer rate. The polymer was applied for the determination of trace RAC through online SPE-HPLC. With a sample loading flow rate of 2 mL/min, the enhancement factor of 516.26 and the LOD (S/N = 3) of 4.6 ng/L were achieved, respectively, and the linear range of the calibration curve was 0.04-18 microg/L with r(2) >0.99. The RAC in pork was determined at three spiked levels (0.5, 1, and 2 ng/g) with recoveries ranging from 55.86 to 67.28%.

Effects of alcohols on CE enantioseparation of basic drugs with native gamma-CD as chiral selector.

The effects of alcohol on the CE enantioseparation of selected basic drugs with gamma-CD as the chiral selector was investigated. The enantioseparation behavior of the analytes with gamma-CD in the absence and presence of different alcohols specifically methanol, ethanol, 2-propanol (IPA), and 2-methyl-2-propanol (TBA), the relationship of enantiomeric resolution (R(s)) values with either hydrophobicity or bulkiness of the alcohols, as well as the effect of these alcohols on interaction of the analytes with gamma-CD were studied. Results showed that hydrophobicity and/or bulkiness of alcohols have an influence on the enantioresolution of most of the analytes based on the relatively high correlation coefficients (R) obtained between R(s) versus log P and between R(s) versus ovality (i.e., parameter to indicate bulkiness of a molecule). Comparison of the values of the average binding constants obtained for each enantiomeric pair in the presence and absence of 5% IPA showed that alcohols can increase, decrease, or give a minimal effect on the analyte-gamma-CD interaction depending on the analyte. Furthermore, the significant enhancement in the enantioresolution of both propranolol and pindolol in the presence of either IPA or TBA led to the baseline enantioresolution of both drugs using 35 mM gamma-CD.

A simple kinetic spectrophotometric method for the determination of isoxsuprine in dosage forms.

A simple and sensitive kinetic method was developed for the determination of isoxsuprine in pharmaceutical preparations. The method is based upon a kinetic investigation of the oxidation reaction of the drug with alkaline potassium permanganate at room temperature for a fixed time of 30 min. The absorbance of the coloured manganate ion was measured at 610 nm. Alternatively, the decrease in the absorbance of potassium permanganate after addition of the drug was measured at 525 nm. The absorbance-concentration plots in both procedures were rectilinear over the range of 0.5-4 microg ml(-1) (r = 0.9998) with a minimum detectability of 0.05 microg ml (-1) (1.48 x 10(-7) M). The different experimental parameters affecting the development and stability of the colours were carefully studied and optimized. The determination of isoxsuprine by the fixed concentration and rate constant methods is also feasible with the calibration equations obtained but the fixed time method has been found to be more applicable. Both procedures were applied to the determination of isoxsuprine in formulations. The results obtained were in good agreement with those obtained using a reference method. The proposed method was also adopted to detect isoxsuprine in spiked human plasma at its therapeutic level of concentration (0.4 microg ml(-1)). A proposal of the reaction pathway was postulated.

Investigations on the stereoselective action of isoxsuprine on alpha- and beta-adrenoceptors in equine common digital artery.

The affinity and functional effects of isoxsuprine enantiomers were investigated to determine the enantiospecificity of the beta-agonistic and alpha-blocking effects. Functional assays on isolated smooth muscle preparations from equine common digital artery were performed to determine the apparent affinity (pD(2)) and intrinsic activity (alpha(E)) of (-)erythro-isoxsuprine (alphaS, betaR, gammaR) and (+)erythro-isoxsuprine (alphaR, betaS, gammaS). The affinity of two enantiomers for the different adrenoceptor types was studied by radioligand binding assays on membrane preparations from the same tissue, using (-)[(3)H]CGP12177 and [(3)H]prazosin. On noradrenaline-precontracted artery preparations (-)isoxsuprine was markedly more potent than (+)isoxsuprine in dilating preparations, indicating that the laevorotatory enantiomer has a very high apparent affinity for alpha-adrenoceptors. Binding studies confirmed that (-)isoxsuprine has a higher affinity than (+)isoxsuprine for alpha-adrenoceptors, while the (+) isomer competes for beta-adrenoceptors with an affinity similar to that of propranolol. As described for other beta-phenylethylamines, the two isoxsuprine enantiomers studied have different efficacies for alpha- and beta-adrenoceptors and the effects of the commercially available mixture of stereoisomers therefore depend on the density and functional importance of the adrenoceptor types present in the tissue studied. 1999 Academic Press.