Inhibition of α-Amylase and α-Glucosidase by New β-Aminopropionamidoxime Derivatives

Pharmaceutical Chemistry Journal - New antidiabetic agents are being sought because of the global problem with diabetes. Amidoxime derivatives are known to have antidiabetic activity....     

P-glycoprotein-mediated transport of digitoxin, α-methyldigoxin and β-acetyldigoxin

Digoxin is a drug with a narrow therapeutic index, which is substrate of the ATP-dependent efflux pump P-glycoprotein. Increased or decreased digoxin plasma concentrations occur in humans due to inhibition or induction of this drug transporter in organs with excretory function such as small intestine, liver and kidneys. Whereas particle size, dissolution rate and lipophilic properties have been identified as determinants for absorption of digitalis glycosides, little is known about P-glycoprotein transport characteristics of digitalis glycosides such as digitoxin, alpha-methyldigoxin, beta-acetyldigoxin and ouabain. Using polarized P-glycoprotein-expressing cell lines we therefore studied whether these compounds are substrates of P-glycoprotein. Polarized transport of digitalis glycosides was assessed in P-glycoprotein-expressing Caco-2 and L-MDR1 cells (LLC-PK1 cells stably transfected with the human MDR1 P-glycoprotein). Inhibition of P-glycoprotein-mediated transport of these compounds in Caco-2 cells was determined using the cyclosporine analogue PSC-833 (valspodar) as inhibitor of P-glycoprotein. No polarized transport was observed for ouabain. However, basal-to-apical transport of digitoxin, alpha-methyldigoxin and beta-acetyldigoxin was greater than apical-to-basal transport in Caco-2 and L-MDR1 cells. In Caco-2 cells net transport rates of these compounds were similar to those of digoxin (digoxin: 16.0+/-4.4%, digitoxin: 15.0+/-3.3%, beta-acetyldigoxin: 16.2+/-1.6%, alpha-methyldigoxin: 13.5+/-4.8%). Furthermore, polarized transport of these compounds could be completely inhibited by 1 microM PSC-833. In summary, these data provide evidence that not only digoxin, but also digitoxin, alpha-methyldigoxin and beta-acetyldigoxin are substrates of P-glycoprotein.     

Self-Assembly of α-Cyclodextrin and β-Cyclodextrin: Identification and Development of Analytical Techniques

Recently, it has been shown that cyclodextrins (CDs) self-assemble in aqueous solutions to form aggregates. Such aggregation can give rise to formation of particulate matter in aqueous solutions. However, the analytical methodology available to detect and quantify these aggregates is still quite inadequate. Here, 5 different methods for evaluation of CD aggregate formation and determination of the critical aggregation concentration are evaluated: osmometry, viscosity, surface tension, dynamic light scattering, and permeability studies. Both the viscosity and surface tension methods applied were inadequate for aggregate detection, whereas the osmometry method can be used to study CD aggregation but with some limitations. Dynamic light scattering has also some limitations although it can be applied to detect CD aggregates and to estimate their hydrodynamic diameter. Overall, permeation studies proved to be the best method to detect and determine critical aggregation concentration. These results suggested that β-cyclodextrin (βCD) has higher tendency to aggregate than α-cyclodextrin (αCD). Filtration of αCD and βCD solutions affected the aggregate size distribution by breaking larger aggregates in to smaller ones that then reassembled to regenerate the larger ones upon storage. The osmolality studies showed that in aqueous αCD and βCD solutions, solute-solute interactions are favored over solute-solvent interactions with consequent CD aggregate formation.     

Antimicrobial activity of pomegranate fruit constituents against drug-resistant Mycobacterium tuberculosis and β-lactamase producing Klebsiella pneumoniae

Abstract

Context: The global surge in multi-drug resistant bacteria and the imminence of tuberculosis pandemic necessitate alternative therapeutic approaches to augment the existing medications. Pomegranate, the fruit of Punica granatum Linn. (Punicaceae), widely recognized for potency against a broad spectrum of bacterial pathogens, deserves further investigation in this respect.

Objective: This study determines the therapeutic potential of pomegranate juice, extracts of non-edible peel prepared with methanol/water, and its four polyphenolic constituents, namely caffeic acid, ellagic acid, epigallocatechin-3-gallate (EGCG) and quercetin, against drug-resistant clinical isolates.

Materials and methods: Phenotypic characterisation of Mycobacterium tuberculosis, extended-spectrum β-lactamase (ESBL) and KPC-type carbapenemase producing Klebsiella pneumoniae was performed by biochemical and molecular methods. Resistance profiles of M. tuberculosis and K. pneumoniae were determined using LJ proportion and Kirby–Bauer methods, respectively. Pomegranate fruit extracts, and the compounds, were evaluated at a dose range of 1024–0.5?µg/mL, and 512–0.25?µg/mL, respectively, to determine minimum inhibitory (MIC) and bactericidal concentrations (MBC) against the drug-resistant isolates by the broth micro-dilution method.

Results: The peel extracts exhibited greater antimycobacterial activity (MIC 64–1024?μg/mL) than the potable juice (MIC 256?-?>?1024?μg/mL). EGCG and quercetin exhibited higher antitubercular (MIC 32–256?μg/mL) and antibacterial (MIC 64–56?μg/mL) potencies than caffeic acid and ellagic acid (MIC 64–512?μg/mL).

Discussion and conclusion: The pomegranate fruit peel and pure constituents were active against a broad panel of M. tuberculosis and β-lactamase producing K. pneumoniae isolates. EGCG and quercetin need further investigation for prospective application against respiratory infections.     

Discovery of cyclic sulfone hydroxyethylamines as potent and selective β-site APP-cleaving enzyme 1 (BACE1) inhibitors: structure-based design and in vivo reduction of amyloid β-peptides

Structure-based design of a series of cyclic hydroxyethylamine BACE1 inhibitors allowed the rational incorporation of prime- and nonprime-side fragments to a central core template without any amide functionality. The core scaffold selection and the structure-activity relationship development were supported by molecular modeling studies and by X-ray analysis of BACE1 complexes with various ligands to expedite the optimization of the series. The direct extension from P1-aryl- and heteroaryl moieties into the S3 binding pocket allowed the enhancement of potency and selectivity over cathepsin D. Restraining the design and synthesis of compounds to a physicochemical property space consistent with central nervous system drugs led to inhibitors with improved blood-brain barrier permeability. Guided by structure-based optimization, we were able to obtain highly potent compounds such as 60p with enzymatic and cellular IC(50) values of 2 and 50 nM, respectively, and with >200-fold selectivity over cathepsin D. Pharmacodynamic studies in APP51/16 transgenic mice at oral doses of 180 μmol/kg demonstrated significant reduction of brain Aβ levels.     

Inclusion complexation of glisentide with α-, β- and γ-cyclodextrins

Complexation of glisentide with α-, β- and γ-cyclodextrin (CD) has been investigated in aqueous solution and in the solid state. Complex formation in solution has been analysed using solubility diagrams and NMR spectroscopy and the interaction in solid state has been studied by X-ray diffractometry, DSC and IR spectroscopy. The thermodynamic parameters, ΔH°, ΔS° and ΔG°, of complexation with β- and γ-CD have been calculated from the temperature dependence of the stability constant. The process has been found to be exothermic and ΔS° is slightly unfavourable. In addition, it has been found that the ionization state of glisentide plays an important role in complexation and the fact that the extent of complexation is greater with β- than with γ-CD has revealed the importance of the cavity size to get an adequate fitting between host and guest molecules. The inclusion of the ortho-substituted aromatic ring of glisentide has been evidenced by NMR spectroscopy. Finally, complexes have been prepared by coprecipitation and kneading methods and it has been found that the former is more suitable to achieve solid-state complexation.     

Optimization of Sample Preparation Conditions for Detecting Trace Amounts of β-Tegafur in α- and β-Tegafur Mixture

We report a semiquantitative method for determining trace amounts (<1%) of thermodynamically stable forms in polymorphic mixtures, focusing on sample preparation effects on solid phase transitions. Tegafur [5-fluoro-1-(oxolan-2-yl)-1,2,3,4-tetrahydropyrimidine-2,4-dione] was used as a model material in this study. The amounts of the thermodynamically stable β tegafur were increased to levels detectable by powder X-ray diffractometry by grinding the samples in a ball mill in the presence of water. The limit of detection for this method was as low as 0.0005% of β tegafur in α and β tegafur mixtures. The amount of β tegafur after sample preparation was found to be proportional to the initial weight fraction of β tegafur. The sum of Langmuir and Cauchy–Lorentz equations was used to describe the change in conversion degree due to the added water volume, where Langmuir equation described water sorption during the sample preparation and Cauchy–Lorentz equation described the grinding efficiency.     

Partial characterization of β-glucosidase, β-xylosidase,and α-l-arabinofuranosidase from Jiangella alba DSM 45237 and their potential in lignocellulose-based biorefining

Jiangella alba DSM 45237 exhibited excellent extracellular β-glucosidase (1.03 ± 0.09 U/mL), β-xylosidase (16.29 ± 0.23 U/mL), and α-l-arabinofuranosidase (7.00 ± 0.09 U/mL) production in the growth media containing 15 g/L wheat straw pretreated with NaOH. The optimum temperature was 40 °C for β-glucosidase and β-xylosidase, whereas it was 50 °C for α-l-arabinofuranosidase. Among them, α-l-arabinofuranosidase was relatively stable at 60 and 70 °C. Enzymes showed maximum activity at pH 8.0. Enzymes, particularly β-glucosidase and α-l-arabinofuranosidase, were able to tolerate NaCl up to a final concentration of 12% (v/w). Among solvents, only ethanol and methanol increased the β-glucosidase activity. The majority of solvents did not significantly affect β-xylosidase activity but increased α-l-arabinofuranosidase activity. Except for phenol, other lignocellulose-derived compounds did not cause a significant activity loss in enzymes. Some of them, such as vanillic acid and acetic acid, have even increased the activity of enzymes. Hydrolysis of pretreated wheat straw using the crude enzyme from J. alba DSM 45237 released 160.9 mg/gds reducing sugars. Analysis of hydrolysis products with thin layer chromatography (TLC) showed that major products were 5C sugars. This is the first report related to the characterization of β-glucosidase, β-xylosidase, and α-l-arabinofuranosidase from J.alba DSM 45237 to date.     

Association of genes coding for the α-4, α-5, β-2 and β-3 subunits of nicotinic receptors with cigarette smoking and nicotine dependence

We assessed whether smoking behavior was associated with nine polymorphisms in genes coding for the nicotinic receptor subunits α-4 (rs1044394, rs1044396, rs2236196 and rs2273504), α-5 (rs16969968), β-2 (rs2072661 and rs4845378) and β-3 (rs4953 and rs6474413). We conducted an Internet survey and collected saliva by mail for DNA and cotinine analyses, in Switzerland in 2003. We conducted DNA analyses for 277 participants and cotinine analyses for 141 current daily smokers. Cotinine levels were higher in carriers of the CC genotype of CHRNA4 rs1044396 (371 ng/ml) than in those with the CT or TT genotypes (275 ng/ml, p = 0.049), a difference of 0.53 standard deviation units. However, this difference was not robust to correction for multiple testing using Bonferroni adjustment. These 9 polymorphisms were not otherwise associated with smoking behavior and nicotine dependence. There were possible associations between the temperament trait novelty seeking and CHRNA4 rs1044396, CHRNA5 rs16969968 and CHRNB2 rs4845378, but these associations were not robust to correction for multiple testing. We conclude that the analysis of polymorphisms in genes coding for four nicotinic acetylcholine receptor subunits (CHRNA4, CHRNA5, CHRNB2 and CHRNB3) and several smoking-related phenotypes revealed no statistically significant association.     

6-Hydroxydopamine pretreatment effects on α- and β-adrenergic receptor adaptation to clorgyline

Summary The effects of 6-hydroxydopamine (6-OHDA) lesions on brain adrenergic receptor adaptation to 21 days of treatment with the selective monoamine oxidase type A (MAO-A) inhibitor clorgyline were studied in rats. 6-OHDA pretreatment effectively blocked the decrease in ₁-, ₂- and -adrenergic receptor densities observed in response to clorgyline treatment. In saline-treated rats, 6-OHDA reduced norepinephrine (NE) to 8% of control levels, modestly reduced dopamine (DA) to 67% of controls, but did not affect serotonin (5HT) levels in the cortex, Clorgyline administration to shams increased NE and 5HT to 239% and 160% of their respective control levels, but did not effect DA levels. 6-OHDA lesions attenuated clorgyline's effect on cortical NE levels but not 5HT. The results suggest that -adrenergic receptor adaptation to MAOI's as with tricyclic antidepressants is a response to an increase in catecholamine receptor occupancy, and that a similar molecular mechanism is responsible for the observed clorgyline induced changes in -adrenergic receptors.     

Synthesis and biological evaluation of α, β-unsaturated ketone as potential antifungal agents

Abstract  With the aim of developing potential antifungals, a series of chalcones incorporating sulfur either as part of a heteroaromatic ring (thiophene) or as a side chain (thiomethyl group) were synthesized and tested for their in vitro activity. Some of the compounds showed appreciable activity against a fluconazole-resistant strain, and could act as new hits for the design of better analogs. Graphical Abstract  Synthesis and biological evaluation of α,β-unsaturated ketone as potential antifungal agentsSeema Bag, S.Ramar, Mariam S. Degani*With an aim to develop new chemical entities with potent antifungal activity against Candida albicans strains, a series of chalcones incorporating sulfur either as part of a heteroaromatic ring (thiophene) or as a side chain (thiomethyl group) were synthesized. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of α- and β-adrenergic receptors linked to human platelet adenylate cyclase

Summary Adrenaline and noradrenaline cause aggregation of human platelets through -adrenergic receptors, whereas isoprenaline through -adrenergic receptors can inhibit aggregation. Either type of adrenergic receptors is coupled to platelet adenylate cyclase. Stimulation and inhibition of adenylate cyclase by -and -adrenergic stimulants, respectively, had been demonstrated in human platelet lysates. These effects were characterized with regard to the effectiveness of various agonists and antagonists.Reduction of platelet adenylate cyclase activity was observed only with L-adrenaline and L-noradrenaline. This inhibitory effect, which was increased in the presence of a -adrenergic blocking agent, was half-maximal at about 1 to 2×10^–6 M adrenaline, and maximal inhibition (by 50–60%) was observed at about 3×10^–5M. Various other catecholamine and imidazoline derivatives that act as -adrenergic agonists in other cell types neither induced aggregation nor affected the enzyme activity.Adrenaline-induced inhibition of platelet adenylate cyclase was prevented by -adrenergic blocking agents. These compounds inhibited the effects of adrenaline on aggregation and on adenylate cyclase with similar efficacies. Dihydrogenated ergot alkaloids were more effective than phentolamine and yohimbine; phenoxybenzamine, tolazoline and azapetine were least effective. Adrenaline-induced inhibition of platelet adenylate cyclase was reversed by phentolamine without apparent lag phase.In the presence of -adrenergic blocking agents, adrenaline was capable of increasing adenylate cyclase activity between 20 and 50%. Only adrenaline and isoprenaline stimulated adenylate cyclase activity; other compounds that stimulate -adrenergic receptors in other cell types, including -adrenergic stimulants, had no effect on the activity of the platelet enzyme. The stimulatory effect of adrenaline was prevented by various -adrenergic blocking agents including pindolol and propranolol. Preferentially -adrenergic receptor blocking agents such as practolol and atenolol were without effect.These findings indicate that the spectrum of compounds capable of exhibiting intrinsic activity through - and -adrenergic receptors of human platelets is very narrow and that either type of platelet adrenergic receptors appears to differ from those found in other cell types.     

Age-related alterations in α1- and β-adrenoceptors mediated responsiveness of rat aorta

Summary We have examined the responsiveness of - and -adrenoceptors in aortae from 1.5, 3, 6 and 24 month old rats. The isometric contraction to phenylephrine was antagonised competitively by prazosin with a pA₂ value of 9.45, suggesting that the receptor is an ₁-adrenoceptor. The potency of phenylephrine was significantly reduced in 24 months old as compared with all younger rats combined. The maximum contraction to phenylephrine was unaltered in 24 month old rats. The maximum contraction to potassium chloride was significantly less than that to phenylephrine only in 1.5 months old rats. In tissues contracted by potassium chloride, isoprenaline produced a marked relaxation in 1.5 months old animals, but there was a progressive loss with increasing age of the -adrenoceptor-mediated relaxation which was markedly reduced by 6 months and abolished in 24 months old. It is concluded that, in the rat aorta, there is a decrease in ₁-adrenoceptor responsiveness in senescence, and a loss of -adrenoceptor-mediated responses in maturation. Send offprint requests to J. R. Docherty at the above address     

Involvement of α2- and β2-adrenoceptors on breast cancer cell proliferation and tumour growth regulation

BACKGROUND AND PURPOSE

β-Adrenoceptors are expressed in human and experimental animal breast cancer cells. However, the effect of the agonists and antagonists reported on cell proliferation and tumour growth was paradoxical, precluding their utilization as possible adjuvant therapy, mainly in the cases of refractory tumours.

EXPERIMENTAL APPROACH

β-Adrenoceptor expression was analysed by immunofluorescence and RT-PCR. Cell proliferation was assessed by [³H]-thymidine incorporation, tumour growth by measuring with a calliper and ERK 1/2 phosphorylation by Western blotting.

KEY RESULTS

β₂-Adrenoceptor expression was confirmed in the mouse and human cells tested. Cell proliferation was increased by adrenaline (by α₂-adrenoceptor action) and decreased in every tested cell line by the β-adrenoceptor agonist isoprenaline and the β₂-adrenoceptor agonist salbutamol. Isoprenaline and salbutamol reduced tumour growth in every tumour tested (mouse C4-HD and CC4-3-HI and human IBH-4, IBH-6 and MDA-MB-231 cell lines growing as xenografts in nude mice). These effects were reversed by the β-adrenoceptor antagonist propranolol. The α₂-adrenoceptor antagonist rauwolscine and the β₂-adrenoceptor agonist salbutamol were equally effective in diminishing tumour growth. ERK 1/2 activation analysed in IBH-4 tumours correlated with tumour growth, with the β-adrenoceptor agonists decreasing its activation. Inhibition of ERK 1/2 phosphorylation in vitro was mainly mediated by the PKA pathway.

CONCLUSIONS AND IMPLICATIONS

In our experimental models, the β-adrenoceptor agonists inhibited breast cancer cell proliferation and tumour growth, probably mediated by inhibition of ERK 1/2 phosphorylation. The β-adrenoceptor agonists were as effective as the α₂-adrenoceptor antagonist rauwolscine, providing possible novel adjuvant treatments for breast cancer.     

Zearalenone and Its Derivatives α-Zearalenol and β-Zearalenol Decontamination by Saccharomyces cerevisiae Strains Isolated from Bovine Forage

Zearalenone (ZEA) and its derivatives are mycotoxins with estrogenic effects on mammals. The biotransformation for ZEA in animals involves the formation of two major metabolites, α- and β-zearalenol (α-ZOL and β-ZOL), which are subsequently conjugated with glucuronic acid. The capability of Saccharomyces cerevisiae strains isolated from silage to eliminate ZEA and its derivatives α-ZOL and β-ZOL was investigated as, also, the mechanisms involved. Strains were grown on Yeast Extract-Peptone-Dextrose medium supplemented with the mycotoxins and their elimination from medium was quantified over time by HPLC-FL. A significant effect on the concentration of ZEA was observed, as all the tested strains were able to eliminate more than 90% of the mycotoxin from the culture medium in two days. The observed elimination was mainly due to ZEA biotransformation into β-ZOL (53%) and α-ZOL (8%) rather than to its adsorption to yeast cells walls. Further, the biotransformation of α-ZOL was not observed but a small amount of β-ZOL (6%) disappeared from culture medium. ZEA biotransformation by yeasts may not be regarded as a full detoxification process because both main end-products are still estrogenic. Nonetheless, it was observed that the biotransformation favors the formation of β-ZOL which is less estrogenic than ZEA and α-ZOL. This metabolic effect is only possible if active strains are used as feed additives and may play a role in the detoxification performance of products with viable S. cerevisiae cells.     

Interactions of the enantiomers of 3-O-methyldobutamine with α- and β-adrenoceptors in vitro

Summary The enantiomers of 3-O-methyldobutamine, a metabolite of dobutamine, were evaluated for their - and -adrenoceptor mediated effects in vitro in a variety of isolated organs and in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed ₁-adrenoceptor agonist activity in isolated guinea pig aorta. However, both enantiomers of 3-O-methyldobutamine were competitive ₁-adrenoceptor antagonists, with the (+)-enantiomer being approximately 10-fold more potent than the (-)-enantiomer as assessed either in guinea pig aorta or by displacement of ³H-prazosin binding from ₁-adrenoceptors in rat cerebral cortex. The ₁-adrenoceptor blocking activity of (+)-3-O-methyldobutamine was relatively potent and corresponded to a pA₂ of 7.33 in guinea pig aorta and a-log K _i of 7.72 in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed ₂-adrenoceptor agonist activity in field-stimulated guinea pig ileum. Although (+)-3-O-methyldobutamine weakly inhibited the twitch response in field-stimulated guinea pig ileum, the response was not blocked by the selective ₂-adrenoceptor antagonist, yohimbine, and was found to result from weak anticholinergic activity (pA₂=5.06). Neither enantiomer of 3-O-methyldobutamine possessed ₁-adrenoceptor agonist activity in guinea pig atria, however the (+)-enantiomer was a weak noncompetitive antagonist at ₁-adrenoceptors. In contrast, both enantiomers of 3-O-methyldobutamine were weak ₂-adrenoceptor agonists in rat uterus, however these weak effects were not highly stereoselective, which was also confirmed in radioligand binding studies. The results of the present study indicate that 3-O-methyldobutamine is a potent and highly selective ₁-adrenoceptor antagonist, with minimal activity at ₂-, ₁- and ₂-adrenoceptors. It is hypothesized that the potent ₁-adrenoceptor antagonist activity of 3-O-methyldobutamine, which resides predominantly in the (+)-enantiomer, may play a role in the hemodynamic effects of dobutamine, by contributing, in part, to the decrease in total peripheral vascular resistance observed following administration of dobutamine.     

Effects of the Aconitum alkaloid mesaconitine in rat hippocampal slices and the involvement of α- and β-adrenoceptors

1. The effects of mesaconitine, the main alkaloid contained in Aconiti tuber, were investigated by use of extracellular recordings of stimulus-evoked population spikes and field excitatory postsynaptic potentials (e.p.s.ps) in the CA1 region of rat hippocampal slices.
2. At a concentration of 10 nM, mesaconitine evoked excitations, which were manifested as an increase in the amplitude of the orthodromic spike and the appearance of multiple spikes following the first postsynaptic spike, without affecting the magnitude of paired-pulse facilitation. The increase in spike amplitude was persistent and was not reversed by up to 90 min of washout. At concentrations of 30 and 100 nM, the alkaloid produced a biphasic effect, that is an excitation followed by an inhibition without having any effect upon the field e.p.s.p. At concentrations above 100 nM, mesaconitine suppressed the orthodromic population spike and the field e.p.s.p.
3. The excitatory effect was also observed when electrical stimulation was stopped completely during the application of mesaconitine (10 nM) and during the first 15 min of washout.
4. The enhancement of the population spike and the appearance of multiple spikes induced by mesaconitine (10–100 nM) were blocked by pretreatment with the β-adrenoceptor antagonists propranolol (1 μM) and timolol (1 μM), whereas the inhibitory effect was blocked by the α-adrenoceptor antagonists yohimbine (1 μM) and phentolamine (10 μM). However, when the β-adrenoceptor antagonist timolol was added 10 min after the application of mesaconitine, it failed to block the long-lasting enhancement of the spike amplitude and the appearance of multiple population spikes.
5. Application of the selective β-adrenoceptor agonist isoprenaline (500 nM) to the hippocampal slices induced an increase in the amplitude of the orthodromic population spike and elicited 2–3 additional spikes. Mesaconitine (10 nM) did not further potentiate this enhancement of the spike amplitude when added after a 15 min pretreatment with isoprenaline.
6. Perfusion of forskolin, which directly activates adenylate cyclase, enhanced the population spike. Mesaconitine had no additional effect when applied after pretreatment with forskolin.
7. It is concluded that the excitatory effects evoked by lower concentrations of the plant alkaloid mesaconitine are mediated by stimulation of β-adrenoceptors and the consequent activation of intracellular processes which lead to the long-lasting changes in excitability.