Catalytic activity and structural changes of catalase in the presence of Levothyroxine and Isoxsuprine hydrochloride.

Two drugs that pregnant women (with hypothyroidism) may use during pregnancy include Isoxsuprine hydrochloride (ISO) and levothyroxine (LEV); ISO to reduce uterine contractions and LEV for the treatment of hypothyroidism. In the current work, we explored the mechanism of binding affinity between the above drugs and antioxidant enzyme Bovine Liver Catalase (BLC). The experimental results confirmed that both drugs could bind with BLC to form drug-BLC complexes but LEV showed a higher binding affinity toward enzyme. The binding constants of LEV-and ISO-BLC were 0.42 × 105 and 0.13 × 104 M-1 at 310 K, respectively. LEV enhanced the catalase activity but the enzymatic activity of BLC reduced gradually in the presence of ISO. Both drugs were able to induce conformational changes in the BLC structure. The results of the molecular docking investigations confirmed the experimental data and showed that the main binding forces in the LEV-BLC and ISO-BLC systems were hydrogen bond and hydrophobic force. The best binding site of both drugs on BLC is located at a cavity among the wrapping domain, N-Terminal arm, and ß-barrel.

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.

Direct MS-MS identification of isoxsuprine-glucuronide in post-administration equine urine.

Isoxsuprine is routinely recovered from enzymatically-hydrolyzed, post-administration urine samples as parent isoxsuprine in equine forensic science. However, the specific identity of the material in horse urine from which isoxsuprine is recovered has never been established, although it has long been assumed to be a glucuronide conjugate (or conjugates) of isoxsuprine. Using ESI/MS/MS positive mode as an analytical tool, urine samples collected 4-8 h after isoxsuprine administration yielded a major peak at m/z 554 that was absent from control samples and resisted fragmentation to daughter ions. Titration of this material with increasing concentrations of sodium acetate yielded m/z peaks consistent with the presence of monosodium and disodium isoxsuprine-glucuronide complexes, suggesting that the starting material was a dipotassium-isoxsuprine-glucuronide complex. Electrospray ionization mass spectrometry negative mode disclosed the presence of a m/z 476 peak that declined following enzymatic hydrolysis and resulted in the concomitant appearance of peaks at m/z 300 and 175. The resulting peaks were consistent with the presence of isoxsuprine (m/z 300) and a glucuronic acid residue (m/z 175). Examination of the daughter ion spectrum of this putative isoxsuprine-glucuronide m/z 476 peak showed overlap of many peaks with those of similar spectra of authentic morphine-3- and morphine-6-glucuronides, suggesting they were derived from glucuronic acid conjugation. These data suggest that isoxsuprine occurs in post-administration urine samples as an isoxsuprine-glucuronide conjugate and also, under some circumstances, as an isoxsuprine-glucuronide-dipotassium complex.

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.

In vitro toxicity and formation of early conjugates in Caco-2 cell line treated with clenbuterol, salbutamol and isoxsuprine.

Caco-2, human intestinal cell line able to differentiate in long-term culture, has been used to assess the cytotoxicity of the beta-agonists clenbuterol, salbutamol and isoxsuprine, also used at high doses to obtain lean meat in food producing animals, and to investigate the eventual in vitro formation of early conjugates of these compounds. For this purpose, the cells have been characterized for the activity of UDP-glucuronyltransferase, which is present and increases in the differentiated cells, and for the beta-receptors' binding characteristics, which are those of beta 1 and beta 2 subtypes. Isoxsuprine was shown to be the most toxic, followed by clenbuterol and salbutamol. Conjugates have been observed after incubation of the cells both with the lowest isoxsuprine and the highest salbutamol concentrations. No conjugates were detected in the case of clenbuterol.

Cardiovascular and pharmacokinetic effects of isoxsuprine in the horse.

Isoxsuprine (0.6 mg/kg) administered IV to 6 standing horses produced substantial, transient decreases in systemic blood pressure, systemic vascular resistance, and stroke volume. It also produced substantial, transient increases in heart rate, cardiac output, and purposeful movement. Plasma concentrations of isoxsuprine peaked soon after the drug was administered IV and then decreased over a 12-hour period in a biexponential manner, with distribution and elimination half-lives of 14 minutes and 2.67 hours, respectively. Total body clearance and steady-state volume of distribution were calculated to be 53.8 ml/min/kg and 10.5 L/kg, respectively. When a recommended therapeutic dosage regimen (0.6 mg/kg 2 times a day, per os) was used in 4 of these horses, changes were not detected. Isoxsuprine was not detected in plasma after the drug was given orally. We conclude that 0.6 mg of isoxsuprine/kg given orally every 12 hours is not likely to produce cardiovascular changes in the resting horse and that this is probably because plasma concentrations are not high enough to do so.

Effects of isoxuprine and nylidrin on adrenoreceptors in rat vas deferens.

The interaction of isoxuprine and nylidrin with alpha 1- and beta 2-adrenoreceptors in rat vas deferens was examined using radioligand binding assays and physiological studies in vitro. Isoxuprine and nylidrin have a greater affinity for binding to alpha 1 (isoxuprine KD = 59 +/- 15 nM; nylidrin KD = 41 +/- 3 nM) than beta 2-(isoxuprine KD = 3,900 +/- 500 nM; nylidrin KD = 900 +/- 50 nM) adrenoreceptors in rat vas deferens. Vas deferens from rats pretreated for 16-24 h with reserpine (3 mg/kg i.p.) were exposed to 10 microM phenoxybenzamine for 15 min to inactivate alpha-adrenoreceptors. Under these conditions high concentrations of both isoxuprine and nylidrin relaxed vas deferens contracted with 55 mM K+, however the relaxation was not blocked by the beta-adrenoreceptor antagonist propranolol (10 microM). Both isoxuprine and nylidrin were potent competitive antagonists of alpha 1-adrenoreceptor mediated contraction of vas deferens. pA2 values for isoxuprine (6.9 +/- .05) and nylidrin (7.1 +/- .08) agreed well with KD values for binding to alpha 1-adrenoreceptors in vas deferens. The greater potency of isoxuprine and nylidrin in inhibiting alpha 1-adrenoreceptors than binding to beta 2-adrenoreceptors or causing nonspecific relaxation suggest that alpha-adrenoreceptor antagonist actions of these drugs may be important in their ability to inhibit smooth muscle tone.

Isoxsuprine infusion in the rat: alterations in maternal, fetal and neonatal glucose homeostasis.

To determine the mechanism of alteration in glucose homeostasis associated with maternal isoxsuprine administration, isoxsuprine or 0.04 M saline was administered intravenously for 3 hours to term pregnant and age-matched virgin rats. Isoxsuprine infusion significantly increased plasma glucose and insulin concentrations and decreased hepatic glycogen stores in both. Compared to rat pups of saline infused mothers, pups of isoxsuprine infused mothers had significantly elevated plasma glucose concentrations for the first 4 hours of life and plasma insulin concentrations for the first two. Plasma glucose concentrations for the offspring of isoxsuprine treated mothers then decreased significantly and remained so until 16 hours of age. Hepatic glycogen concentrations were significantly less in rat pups of isoxsuprine treated mothers at birth and for the first 4 hours of life. In a limited number of studies, isoxsuprine was present at birth in substantial quantities (80-85% of maternal levels) in the plasma of rat pups of isoxsuprine infused mothers. These data suggest that maternal isoxsuprine therapy mobilizes hepatic glycogen and results in maternal hyperlgycemia. Maternal isoxsuprine infusion may directly deplete fetal hepatic glycogen and result in transient fetal and neonatal hyperglycemia. the in utero depletion of glycogen and possibly, the early stimulation of insulin production may be responsible for the later significant decreases in plasma glucose in the offspring of isoxsuprine treated mothers.

Non-release of lactic acid from anaerobic swimming muscle of plaice Pleuronectes platessa L.: a stress reaction.

1. Plaice caught by trawl net and plaice exercised in laboratory tanks all show high levels of lactic acid (33--44 mmol/kg) in the anaerobic swimming muscle. During exhausting exercise 2 moles of lactate are formed from 1 mole of glycogen glucose. After an 8 h rest 50--80% of the muscle glycogen is restored. 2. Blood lactate levels remain low (0.5--2 mmol/l) in the majority of plaice caught by trawl. In a small number of plaice, peak levels over 5 mmol/l are reached 2--4 h after capture. Low blood lactate levels could be guaranteed in all fish exercised 24 h after the stress of capture and in tank-adapted fish exercised and injected with the beta-adrenergic stimulating drug, isoxsuprine hydrochloride. The blood lactate in plaice, tank-adapted for more than 8 days and then exercised, may reach peak levels up to 5 mmol/l 2--4 h later. 3. High blood lactate levels were obtained by injecting the beta-adrenergic block propranolol to stressed exercised fish. The alpha-adrenergic block did not have this effect. All plaice with blood lactate levels reaching 5--12 mmol/l died. 4. The results indicate that the muscle cells regulate the release or nonrelease of their lactate load to the blood stream and increases in the blood circulating to the muscle do not influence this release. The non-release mechanism may be actived by a catecholamine circulated in the blood stream following a stress.