A set of multiplex PCRs for genotypic detection of extended-spectrum β-lactamases, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases

Worldwide, resistance of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to β-lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum β-lactamases (ESBLs) are most widely disseminated, other β-lactamase families have also recently emerged, such as plasmid-mediated AmpC β-lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent β-lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC β-lactamases and OXA β-lactamases.     

Intestinal Absorption of Acyclovir β-Glucoside: Comparative Study with Acyclovir,Guanosine, and Kinetin β-Glucoside

Purpose. To characterize the intestinal absorption of a -glucose conjugate of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV) and compare it to ACV and its analogues in terms of stability and transport by Na⁺/glucose cotransporter (SGLT1). Methods. ACVglc was enzymatically synthesized using cellulase. Intestinal absorption experiments were performed with rat everted small intestine. Conformation of the glucose moiety was analyzed by NMR spectroscopy. Results. The ACVglc was stable on the mucosal side, and was transported to the serosal side in all regions of the small intestine. However, significant contribution of SGLT1 to the transport of ACVglc was not observed. NMR spectroscopic analysis indicated that the glucose conformation of ACVglc was the ⁴C₁ chair form, identical to (-glucose or SGLT1-transportable -glucosides reported previously. Therefore, other factors such as molecular size and charge due to aglycone may cause no transport of ACVglc by SGLT1. On the other hand, the hydrophilicity of ACVglc was much higher than of ACV, suggesting water solubility-derived improvement of intestinal absorption of ACV. Conclusions. ACVglc is stable and absorbable, but it is not transported by SGLT1. No involvement of SGLT1 in the ACVglc transport is not due to glucose conformation.     

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.     

Review of synthesis, biological assay and QSAR studies of β-secretase inhibitors

Alzheimer's disease (AD) is highly complex. While several pathologies characterize this disease, amyloid plaques, composed of the β-amyloid peptide, are hallmark neuropathological lesions in Alzheimer's disease brain. Indeed, a wealth of evidence suggests that β-amyloid is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. The BACE-1 enzyme is essential for the generation of β-amyloid. BACE-1 knockout mice do not produce β-amyloid and are free from Alzheimer's associated pathologies, including neuronal loss and certain memory deficits. The fact that BACE-1 initiates the formation of β-amyloid, and the observation that BACE-1 levels are elevated in this disease provide direct and compelling reasons to develop therapies directed at BACE-1 inhibition, thus reducing β-amyloid and its associated toxicities. In this sense, quantitative structure-activity relationships (QSAR) could play an important role in studying these β-secretase inhibitors. QSAR models are necessary in order to guide the β-secretase synthesis. This work is aimed at reviewing different design and synthesis and computational studies for a very large and heterogeneous series of β-secretase inhibitors. First, we review design, synthesis, and Biological assay of β-secretase inhibitors. Next, we review 2D QSAR, 3D QSAR, CoMFA, CoMSIA and Docking with different compounds to find out the structural requirements. Next, we review QSAR studies using the method of Linear Discriminant Analysis (LDA) in order to understand the essential structural requirement for receptor binding for β- secretase inhibitors.     

Effects of naloxone, β-endorphin and ACTH on acquisition of schedule-induced polydipsia

A series of three experiments examined the possible involvement of endogenous opioid peptides in the development of schedule-induced polydipsia in rats. Repeated pretraining treatment with 2 mg/kg naloxone impaired acquisition of schedule-induced polydipsia, whereas the same treatment injected after training increased drinking. This later effect was time dependent, since a 30-min delay in the injection of naloxone resulted in a disappearance of its effect. Post-training injections of 10 g/kg -endorphin or ACTH delayed the development of drinking. These findings are consistent with the hypothesis that endogenous opioid peptides modulate the development of schedule-induced polydipsia.     

Pharmacokinetics of amosulalol,an α, β-adrenoceptor blocker,in rats,dogs and monkeys

1. The pharmacokinetics of an α,β-adrenoceptor blocker, amosulalol hydrochloride, were studied after i.v. and oral administration to rats, dogs and monkeys.

2. After an i.v. dose (1 mg/kg), the plasma concentration-time curve fitted a two-compartment open model with terminal half-lives of 2-5 h in rats, 21 h in dogs and 1-8 h in monkeys. The order of plasma clearances for amosulalol was: rats > dogs > monkeys.

3. After oral administration, the maximum plasma concentration was obtained at 0-5-1 h in rats (10-100mg/kg) and dogs (3-30mg/kg), and at l-7-2-7h in monkeys (3-10 mg/kg). A linear relationship between the area under the plasma concentration-time curve and dose administered was obtained for all three species. The systemic availabilities of the drug in rats, dogs and monkeys were 22-31%, 51-59% and 57-66%, respectively.

4. After repeated oral administration (10 mg/kg) to dogs for 15 days, the pharmaco-kinetic parameters did not differ significantly from those on the first day.     

Transdermal delivery of β-blockers

β-Adrenoceptor blocking drugs (β-blockers) are one of the most frequently used class of cardiovascular drugs that are mainly used in conventional dosage forms., which have their own limitations including hepatic first-pass metabolism, high incidence of adverse effects due to variable absorption profiles, higher frequency of administration and poor patient compliance. Essentially, attempts have been made to develop novel drug delivery systems for β-blockers, including transdermal delivery systems, to circumvent the drawbacks of conventional drug delivery. However, so far none of the β-blocker drugs have been marketed as transdermal delivery systems. Nevertheless, there have been noteworthy research endeavours worldwide at the laboratory level to investigate the skin permeation and to develop transdermal formulations of β-blockers including: propranolol, metoprolol, atenolol, timolol, levobunolol, bupranolol, bopindolol, mepindolol, sotalol, labetolol, pindolol, acebutolol and oxprenolol. Innovative research exploiting penetration-enhancing strategies, such as iontophoresis, electroporation, microneedles and sonophoresis, holds promise for the successful use of these drugs as consumer-friendly transdermal dosage forms in clinical practice. This paper presents an overview of the transdermal research on this important class of drugs.     

Pharmacogenetic characterization of indacaterol,a novel β2-adrenoceptor agonist

Background and purpose:

Indacaterol is a novel β₂-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human β₂-adrenoceptor and in human primary airway smooth muscle (ASM) cells.

Experimental approach:

Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human β₂-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3′,5′-cyclic-monophosphate production was used as an outcome measure.

Key results:

In both the low- and high-expression recombinant GEVT ‘wild type’ cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the β₂-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3′,5′-cyclic-monophosphate in response to β₂-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol.

Conclusions and implications:

These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of β₂-adrenoceptors.     

Clinical pharmacology of a new β-adrenoceptor blocking drug,befunolol

Summary Repeated doses of a new -adrenoceptor blocking agent, befunolol, were administered orally to adult male volunteers for a cross-over comparison with propranolol. The -adrenoceptor blocking activity of befunolol was greater than that of propranolol when assessed by the percentage reduction in exercise-induced tachycardia. The elimination half-life of drug was significantly prolonged on repeated administration of propranolol, but not of befunolol. The percentage reduction in exercise-induced tachycardia was highly correlated with the log plasma level of each drug. Both drugs produced a significant reduction in pre-exercise systolic and diastolic blood pressure, and significant attenuation of exercise-induced rise in systolic blood pressure.     

Effects of a hydroxylated metabolite of the β-adrenoreceptor antagonist,carvedilol, on post-ischaemic splanchnic tissue injury

1. Reactive oxygen species have been demonstrated to play a critical role in post-ischaemic tissue injury. The present experiment was designed to evaluate the effects of SB 211475, a hydroxylated metabolite of the new β-adrenoceptor antagonist, carvedilol, on rat splanchnic ischaemia (SI, 60 min) and reperfusion(R)-induced shock and tissue injury.
2. Administration of SB 211475 two min before R attenuated SI/R injury in a dose-dependent manner. At doses of 0.5 mg kg⁻¹ and 1.0 mg kg⁻¹, SB 211475 exerted significant anti-shock and endothelial protective effects, characterized by prolonged survival times, increased survival rates, attenuated increases in tissue myeloperoxidase activity and haematocrits, and preserved endothelium-dependent vasorelaxation.
3. Administration of 1 mg kg⁻¹ carvedilol attenuated shock-induced tissue injury and endothelial dysfunction. However, administration of 0.5 mg kg⁻¹ carvedilol had no protective effects on post-ischaemic tissue injury.
4. Previous studies have shown that SB 211475 has virtually no β-blocking activity but possesses more potent antioxidant activity than carvedilol. In the present study, SB 211475 exerted more potent protective effects than the parent compound, suggesting that this metabolite of carvedilol is superior to carvedilol with regard to its protection against post-ischaemia tissue injury.

     

Levels and circadian rhythmicity of plasma ACTH,cortisol, and β-endorphin as a function of family history of alcoholism

Rationale Individuals with a family history of alcoholism may present a dysfunction in the activity of the hypothalamic–pituitary–adrenal (HPA) axis that predates the development of alcoholism. Objective The present study investigated the hypothesis that this HPA-axis dysfunction is associated with alterations in the pattern of the circadian (24 h) secretions of adrenal corticotropic hormone (ACTH), cortisol, and β-endorphin. Methods Men with [high risk (HR)] or without [low risk (LR)] family history of alcoholism participated in the study. Blood samples were drawn every 30 min for 24 h for estimation of the plasma hormone levels. Participants ingested meals at predetermined intervals and filled out mood questionnaires prior to the placement of the catheter and 1 h after each meal. Results The circadian peaks for β-endorphin, ACTH, and cortisol occurred between 0800 and 0830 hours in both LR and HR participants. The plasma ACTH and β-endorphin concentrations were lower in HR than LR participants, while the plasma cortisol concentrations were similar between HR and LR participants. For each hormone, the total 24-h secretion was estimated from the area under the 24-h time–concentration curve (AUC). For ACTH and β-endorphin, but not the cortisol, AUC were lower in HR than LR participants. LR participants reported being more nervous than HR participants. For the LR participants, but not HR participants, the initial mood ratings of “nervous” were positively correlated with the initial plasma cortisol and β-endorphin concentrations as well as with the cortisol and β-endorphin AUC. Conclusions HR participants presented lower plasma concentrations as well as lower AUC for β-endorphin and ACTH but not for cortisol. This suggests a dysfunction of the HPA-axis in HR participants that predates the development of alcoholism and a dissociation between plasma ACTH and cortisol levels as a function of family history of alcoholism.     

Hepatoprotective effect of lactic acid bacteria, inhibitors of β-glucuronidase production against intestinal microflora

The hepatoprotective activity of lactic acid bacteria (Lactobacillus brevis HY7401, Lactobacillus acidophilus CSG and Bifidobacterium longum HY8001), which inhibited beta-glucuronidase productivity of intestinal microflora, on t-BHP- or CCl4-induced hepatotoxicity of mice were evaluated. These oral administration of lactic acid bacteria lowered beta-glucuronidase production of intestinal microflora as well as Escherichia coli HGU-3. When lactic acid bacteria at a dose of 0.5 or 2 g (wet weight)/kg was orally administered on CCl4-induced liver injury in mice, these bacteria significantly inhibited the increase of plasma alanine transferase and aspartate transferase activities by 17-57% and 57-66% of the CCl4 control group, respectively. These lactic acid bacteria also showed the potent hepatoprotective effect against t-BHP-induced liver injury in mice. The inhibitory effects of these lactic acid bacteria were more potent than that of dimethyl diphenyl bicarboxylate (DDB), which have been used as a commercial hepatoprotective agent. Among these lactic acid bacteria, L. acidophilus CSG exhibited the most potent hepatoprotective effect. Based on these findings, we insist that an inhibitor of beta-glucuronidase production in intestine, such as lactic acid bacteria, may be hepatoprotective.     

The Development of β-Lactam Antibiotics in Response to the Evolution of β-Lactamases

beta-Lactam antibiotics, viz., penicillin, penicillin derivatives, cephalosporins, cephamycins, carbapenems, monobactams. and monocarbams, are the most widely used of all antimicrobial classes by virtue of their high efficacy and specificity and the availability of several derivatives. The expression of one or several beta-lactamases (beta-lactam antibiotic-inactivating enzymes) represents the most widespread and the most clinically relevant resistance mechanism to these antibiotics. The development of beta-lactam antibiotics has thus been a continuous battle of the design of new compounds to withstand inactivation by the ever-increasing diversity of beta-lactamases. This article traces antibiotic development in response to the evolution of beta-lactamases.     

Pharmaceutical evaluation of hydroxyalkyl ethers of β-cyclodextrins

Hydroxyalkylated β-cyclodextrins (HA-β-CyDs), 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD), and hydroxyethyl-β-cyclodextrin (HE-β-CyD), were prepared and their physicochemical and biological properties and solubilizing abilities were studied and compared with those of parent β-cyclodextrin (β-CyD). HA-β-CyDs had much higher aqueous solubility ( > 50 w/v%) and were less hygroscopic than the parent β-CyD. Their surface activities were between those of β CyD and alkylated-β-cyclodextrins and were increased proportionately to their average degrees of substitution. The hemolytic activity (human erythrocytes) and local irritancy (rabbit muscle) of these compounds, and particularly of HE-β-CyD, were considerably less than those of natural cyclodextrin or dimethyl-β-cyclodextrin (DM-β-CyD). In contrast to surface activity, the hemolytic activity of HA-β-CyD decreased with the degree of substitution; possibly the difference in their ability to dissolve membrane components may be the reason. HA-β-CyDs were found to be powerful solubilizers of several drugs and no crystalline complexes were precipitated at high concentrations of solubilizer, a phenomenon which is often observed when β-CyD is used. HP-β-CyDs were somewhat better solubilizers than HE-β-CyDs and the preparations with the lower degrees of substitution were again better than those with the higher ones. The above data suggest that HA-β-CyDs are safer and more effective solubilizers for poorly water-soluble drugs than the parent cyclodextrin.     

Anti-inflammatory potential of β-amyrin,a triterpenoid isolated from Costus igneus

Costus igneus, common name Fiery Costus or Spiral Flag, is a species of herbaceous plant in the Costaceae family. It is cultivated in India for its use in traditional medicine especially for diabetes. The present study was carried out to determine the mechanism of anti-inflammatory action of β-amyrin isolated from the leaves of Costus igneus (C. igneus) using carrageenan-induced rat model and LPS-induced human peripheral blood mononuclear cells (hPBMCs) in vitro model. The differential fractionation of leaves of Costus igneus showed maximum percentage inhibition of paw edema at a dose of 100 mg/kg body weight in methanolic extract (MEC). MEC elicited significant anti-inflammatory effect by inhibiting cyclooxygenase (COX), lipoxygenase (LOX), myeloperoxidase (MPO) and nitric oxide synthase (NOS) activities in monocytes when compared to carrageenan control. The effect of MEC was more pronounced than standard drug Diclofenac (20 mg/kg body weight). After fractionation of MEC using various solvents such as chloroform, hexane, ethyl acetate and butanol, the mechanism of anti-inflammatory effect of chloroform extract (CEC) of MEC was evaluated since it showed maximum beneficial effect at a dose of 50 mg/kg BW Treatment of carrageenan-induced rats with CEC exerted significantly decreased COX-2, MPO, and NOS activities when compared to carrageenan-induced rats. By the partial purification of CEC by liquid–liquid partition chromatography, TLC, mass, IR and NMR spectroscopy, the active component β-amyrin was isolated. Significant decrease in edema was observed by the administration of β-amyrin in a dose-dependent manner and 100 µg of β-amyrin showed 97 % in carrageenan-induced paw edema in rats. Treatment with β-amyrin significantly inhibited PGE2, IL-6 secretion, and NF-κB activation in a concentration-dependent manner on LPS-induced hPBMCs. Thus, β-amyrin, an active component isolated from C. igneus, serves as a promising and expanding platform for treatment of various inflammatory disorders.     

Effects of age,phenobarbital, β-naphthoflavone and dexamethasone on rat hepatic heme oxygenase

There is a wide range of change in both microsomal heme oxygenase activity and cytochrome P-450 level in the livers of rats of various ages. We tried to investigate the phases of heme oxygenase activity, both spontaneous and caused by typical MFO inducers in the lifetime of the rat. Wistar male rats aged 0.5, 1, 2, 4, 8, 12, 20 and 28 months received phenobarbital (50 mg/kg) twice, 3 and 2 days before being killed. ß-Naphthoflavone and dexamethasone were given three times: 3, 2 and 1 day before decapitation 20 mg/kg and 10 mg/kg, respectively). The highest heme oxygenase activity is observed in intact 2-week-old animals (1.16±0.038 nM/h per mg protein). Before maturity this activity decreases slightly up to the 2nd month of life. Then it stabilizes and remains virtually unchanged till the 8th month of life (1.02±0.03). Afterwards HO activity tends to increase until the 28th month of life (1.10±0.06), but does not reach the level observed in the 2-week-old animals. We have found that some typical MFO inducers can modify HO activy. While phenobarbital stimulates HO activity only in premature animals (1.42±0.056; 1.30±0.059 and 1.13±0.035, respectively in 0.5-, 1- and 2-month-old animals), ß-naphthoflavone enhances HO activity in all the groups studied. Dexamethasone, as a physiological inducer of the MFO system, modifies HO activity very characteristically. It induces this activity until the 2nd month of life and then its inducibility appears to remain unchanged. Correlations between HO activity, ALAS activity and cytochrome P-450 levels still need to be investigated to elucidate these problems.     

Synthesis,Characterization, and Evaluation of the Local Irritant Action of an Ibuprofen - β-Cyclodextrin Inclusion Complex

Pharmaceutical Chemistry Journal -     

Aqueous stability of leuprolide acetate: effect of temperature,dissolved oxygen,pH and complexation with β-cyclodextrin

In the present research, the aqueous stability of leuprolide acetate (LA) in phosphate buffered saline (PBS) medium was studied (pH?=?2.0–7.4). For this purpose, the effect of temperature, dissolved oxygen and pH on the stability of LA during 35 days was investigated. Results showed that the aqueous stability of LA was higher at low temperatures. Degassing of the PBS medium partially increased the stability of LA at 4?°C, while did not change at 37?°C. The degradation of LA was accelerated at lower pH values. In addition, complexes of LA with different portions of β-cyclodextrin (β-CD) were prepared through freeze-drying procedure and characterized by Fourier transform infrared (FTIR) and differential scanning calorimetry (DSC) analyses. Studying their aqueous stability at various pH values (2.0–7.4) showed LA/β-CD complexes exhibited higher stability when compared with LA at all pH values. The stability of complexes was also improved by increasing the portion of LA/β-CD up to 1/10.     

Antibacterial, antifungal and antioxidant activity of some new water-soluble β-diketones

A new series of eight compounds 1-(4′-O-β-d-glucopyranosyloxy-2′-hydroxy-5′-substituted phenyl)-3-heteroaryl-propane-1,3-diones (DKG) were prepared by the interaction of α-acetobromoglucose with 1-(2′,4′-dihydroxy-5′-substituted phenyl)-3-heteroaryl-propane-1,3-diones under anhydrous condition at lower temperature. The structures of these O-β-d-glucopyranosides were established on the basis of chemical, elemental, and spectral analyses. Further, the compounds were tested for their antibacterial, antifungal, and antioxidant properties. A correlation of structure and activities relationship of these compounds with respect to molecular modeling, Lipinski rule of five, drug-likeness, toxicity profiles, and other physico-chemical properties of drugs are described and verified experimentally.