CK2 inhibitors increase the sensitivity of HSV-1 to interferon-β

Herpes simplex virus type 1 (HSV-1) requires the activities of cellular kinases for efficient replication. The host kinase, CK2, has been shown or is predicted to modify several HSV-1 proteins and has been proposed to affect one or more steps in the viral life cycle. Furthermore, potential cellular and viral substrates of CK2 are involved in antiviral pathways and viral counter-defenses, respectively, suggesting that CK2 regulates these processes. Consequently, we tested whether pharmacological inhibitors of CK2 impaired HSV-1 replication, either alone or in combination with the cellular antiviral factor, interferon-β (IFN-β). Our results indicate that the use of CK2 inhibitors results in a minor reduction in HSV-1 replication but enhanced the inhibitory effect of IFN-β on replication. This effect was dependent on the HSV-1 E3 ubiquitin ligase, infected cell protein 0 (ICP0), which impairs several host antiviral responses, including that produced by IFN-β. Inhibitors of CK2 did not, however, impede the ability of ICP0 to induce the degradation of two cellular targets: the promyelocytic leukemia protein (PML) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Notably, this effect was only apparent for HSV-1, as the CK2 inhibitors did not enhance the antiviral effect of IFN-β on either vesicular stomatitis virus or adenovirus type 5. Thus, our data suggest that the activity of CK2 is required for an early function during viral infection that assists the growth of HSV-1 in IFN-β-treated cells.     

Hepatoprotective effects and HSV-1 activity of the hydroethanolic extract of Cecropia glaziovii (embaúba-vermelha) against acyclovir-resistant strain

Context: Cecropia glaziovii Snethl. (Cecropiaceae), commonly known as “embaúba-vermelha”, is widely distributed throughout Latin America and has been reported in Brazilian folk medicine to treat cough, asthma, high blood pressure and inflammation.

Objective: Investigate the hepatoprotective properties of crude hydroethanolic extract of C. glaziovii as well as its in vitro antioxidant and antiviral (HSV-1 acyclovir resistant strain) activities.

Materials and methods: The hepatoprotective effect, the antioxidant properties and antiviral activity of crude hydroethanol extract (RCE40) from C. glaziovii leaves were evaluated by carbon-tetrachloride (CCl₄)-induced hepatotoxicity, by TBARS (thiobarbituric acid reactive species) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays, respectively.

Results: The RCE40 extract (20?mg/kg) inhibited lipid peroxidation on liver in post injury treatment and decreased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In addition, in this protocol the RCE40 (20?mg/kg) enhanced the activity of hepatic enzymes (SOD/CAT) which are involved in combating reactive oxygen species (ROS), suggesting that it possesses the capacity to attenuate the CCl₄-induced liver damage. Moreover the RCE40 (20?mg/kg) inhibited TBARS formation induced by several different inductors of oxidative stress showing significant antioxidant activity, including physiologically relevant concentration, as low as 2 µg/mL. Concerning antiviral activity, the RCE40 was effective against herpes simplex virus type 1 replication (29R acyclovir resistant strain) with EC₅₀?=?40 µg/mL and selective index (SI)?=?50.

Discussion and conclusion: These results indicate that C. glaziovii could be a good source of antioxidant and anti-HSV-1 lead compounds.     

Differential sensitivity of TREK–1, TREK–2 and TRAAK background potassium channels to the polycationic dye ruthenium red

Background and Purpose

Pharmacological separation of the background potassium currents of closely related K_2P channels is a challenging problem. We previously demonstrated that ruthenium red (RR) inhibits TASK-3 (K_2P9.1), but not TASK-1 (K_2P3.1) channels. RR has been extensively used to distinguish between TASK currents in native cells. In the present study, we systematically investigate the RR sensitivity of a more comprehensive set of K_2P channels.

Experimental Approach

K⁺ currents were measured by two-electrode voltage clamp in Xenopus oocytes and by whole-cell patch clamp in mouse dorsal root ganglion (DRG) neurons.

Key Results

RR differentiates between two closely related members of the TREK subfamily. TREK-2 (K_2P10.1) proved to be highly sensitive to RR (IC₅₀ = 0.2 μM), whereas TREK-1 (K_2P2.1) was not affected by the compound. We identified aspartate 135 (D135) as the target of the inhibitor in mouse TREK-2c. D135 lines the wall of the extracellular ion pathway (EIP), a tunnel structure through the extracellular cap characteristic for K_2P channels. TREK-1 contains isoleucine in the corresponding position. The mutation of this isoleucine (I110D) rendered TREK-1 sensitive to RR. The third member of the TREK subfamily, TRAAK (K_2P4.1) was more potently inhibited by ruthenium violet, a contaminant in some RR preparations, than by RR. DRG neurons predominantly express TREK-2 and RR-resistant TREK-1 and TRESK (K_2P18.1) background K⁺ channels. We detected the RR-sensitive leak K⁺ current component in DRG neurons.

Conclusions and Implications

We propose that RR may be useful for distinguishing TREK-2 (K_2P10.1) from TREK-1 (K_2P2.1) and other RR-resistant K_2P channels in native cells.     

Development of noncytotoxic PLGA nanoparticles to improve the effect of a new inhibitor of p53–MDM2 interaction

One possible approach to overcome solubility complications and enhance the biological activity of drugs is their incorporation into drug delivery systems. Within this scope, several nanosphere and nanocapsule formulations of a new inhibitor of p53–MDM2 interaction (xanthone 1) were developed and their physicochemical properties analyzed. Through the investigation of the effect of several empty nanoparticles on the growth of MCF-7 cells, it was possible to observe that four out of five formulations were cytotoxic and that some correlations between the toxic potential of these polymeric nanoparticles and their properties/composition could be extrapolated. One empty formulation of nanocapsules developed by emulsification/solvent evaporation and containing PLGA, PVA and Mygliol^® 812 was found to be noncytotoxic to this cell line. The corresponding compound 1-loaded nanocapsules showed an incorporation efficiency of 77% and revealed to be more potent than the free drug against cell growth inhibition, which may be related to the enhancement in its intracellular delivery. In an integrative study, the intracellular uptake of nanocapsules was confirmed using fluorescent 6-coumarin and well as compound 1 release from nanocapsules. Overall, it was possible to enhance the effect of the hit inhibitor of p53–MDM2 interaction through the development of suitable noncytotoxic polymeric nanoparticles.     

Inhibitory effect and structure–activity relationship of some Biginelli-type pyrimidines against HSV-1

Inevitable emergence of multi-drug resistant strains to current available drugs makes an impetus for finding new therapeutic agents against herpes simplex virus (HSV). In this study, a series of novel derivatives of Biginelli-type pyrimidines were evaluated as potential anti-HSV-1 compounds by plaque reduction method. The cellular toxicity was assessed by XTT proliferation assay. The time course of anti-HSV activity of the most active compound was studied to show the anti-viral effect in various intervals of replication cycle. Compounds 2, 6, 8, 11, 12, 17, 18, 20, and 40 had the highest activity for inhibition of HSV. Compound 40 inhibited HSV replication with IC₅₀ of 0.9 μmol/l and had CC₅₀ of up to 100 μmol/l. This compound was a noteworthy inhibitor against HSV with TI value of 111. Compound 20 also showed considerable inhibitory activity with IC₅₀ of 1.8 μmol/l. Result of time-of-addition study showed that compound 40 inhibits HSV replication at a stage after entry of virions to the target cells. Analysis of structure of the studied compounds demonstrated clear relationships with their anti-HSV effects. Some of the compounds seem to be promising candidates for future anti-HSV drug discovery researches. Structural manipulation based on the obtained structure–activity relationships would propose some new leads for anti-HSV drug discovery programs.     

3-Aminobenzamide – a PARP inhibitor enhances the sensitivity of peripheral blood micronucleus and comet assays in mice

Context: DNA repair is an essential outcome of DNA damage, which may compromise the end point of various in vitro and in vivo test systems of the genotoxicity evaluation. poly(ADP-ribose) polymerase (PARP) enzymes have an essential role in DNA repair. Here, we investigated the effect of 3-AB, a PARP inhibitor on the sensitivity of comet and PBMN assays.

Objective: This study aimed to enhance the sensitivity of the comet and peripheral blood micronucleus (PBMN) assays using 3-aminobenzamide (3-AB), a well-characterized PARP inhibitor.

Materials and methods: Cyclophosphamide (CP, 50?mg/kg), 5-flourouracil (5-FU, 25?mg/kg), zidovudine (AZT, 400?mg/kg) and furosemide (FUR, 60?mg/kg) were selected as genotoxins. 3-AB was given every 8?h with the first dose given 2?h before the genotoxin treatment. For the PBMN assay, small amount of blood was taken from the tail tip of each animal and smears were prepared. The comet assay was performed in PBL, bone marrow and liver.

Results: In the comet as well as PBMN assay, 3-AB pre-treatment enhanced the extent of DNA damage in all the combination groups (3-AB?+?CP, 3-AB?+?5-FU and 3-AB?+?AZT) compared to CP, 5-FU and AZT per se. 3-AB also enhanced the DNA damage caused by FUR in the bone marrow and liver.

Discussion: This study results clearly demonstrate that the pretreatment with 3-AB (30?mg/kg) significantly enhances the sensitivity of the PBMN and comet assays. This model may be useful for the detection of marginally active DNA damaging agents.     

A review of the clinical pharmacokinetics and metabolism of the α1-adrenoceptor antagonist indoramin

1. The α₁-adrenoceptor antagonist indoramin is rapidly and extensively absorbed after oral administration, but with only low to moderate bioavailability (8-24% median) from the tablet (Baratol®). Although plasma protein binding is high (72-86%), the drug is widely distributed into tissues (with median V_z 6.3.7.71/kg after i.v. dosage).

2. Elimination of indoramin is rapid in most healthy volunteers, with median plasma clearances of 18-26ml/min per kg, after i.v. dosage. Elimination occurs principally by metabolism, the major route being indole 6-hydroxylation, followed by sulphate conjugation of 6-hydroxyindoramin. The faecal route of excretion predominates (45-50% of dose), with a further 35-40% in the urine.

3. Extensive variation in single-dose oral pharmacokinetics of indoramin is due largely to the existence of a poor metabolizer phenotype which co-segregates with that of debrisoquine.

4. On repeated administration (37.5 mg twice daily) to healthy volunteers, plasma concentrations of indoramin accumulate 3-4-fold above those anticipated from single-dose kinetics. However, steady state is achieved within the first week of dosing.

5. The pharmacokinetics of indoramin are substantially altered in the elderly. The oral AUC for a 50mg dose is increased approx. 5-fold and the t_1/2 2-5-fold.

6. Cirrhotic liver disease enhances bioavailability and decreases clearance, approx. 2-fold in each case for single oral and i.v. doses of 50mg and 0.15mg/kg respectively.

7. After oral indoramin C_max and AUC are both raised (58% and 25%, respectively, for a 50 mg dose) by co-ingested ethanol (0.5 g/kg). After i.v. indoramin, kinetics are unaffected by alcohol, but indoramin (0.175 mg/kg) slightly increases (26%) blood ethanol concentrations during the first hour after dosing.

8. The pharmacodynamics of indoramin appear to be related to the combined pharmacokinetics of the drug and its 6-hydroxylated metabolite, which contributes to the antihypertensive effect.     

Translation and validation of a tool to assess the impact of clinical pharmacists’ interventions

International Journal of Clinical Pharmacy - Background The tool CLEO in French language is designed for estimating the potential relevance of pharmacists’ interventions (PIs) in three...     

Gestational exposure to the organophosphate chlorpyrifos alters social–emotional behaviour and impairs responsiveness to the serotonin transporter inhibitor fluvoxamine in mice

Background

The organophosphate chlorpyrifos (CPF) is a pesticide largely used worldwide. Studies from animal models indicate that CPF exposure during development at low doses can target different neurotransmitter systems in the absence of overt cholinergic effects.

Methods

Late gestational exposure (gestational days 14–17) to CPF at the dose of 6 mg/kg was evaluated in CD-1 mice at adulthood. Neurobehavioural effects likely involving serotonin (5-hydroxytryptamine, 5HT) transmission were assessed both in males and females, through the light–dark exploration test to assess CPF effects on anxiety profiles and the forced swimming test to evaluate the response to the 5HT transporter (5HTT) inhibitor fluvoxamine (30 mg/kg). In females only, we evaluated the effects of gestational exposure to CPF on maternal aggression, under basal condition or after injection of fluvoxamine.

Results

Gestational CPF exposure increased anxiety levels only in female mice, as shown by the augmented thigmotaxis behaviour and the lower latency to enter in the dark compartment. In the forced swimming test, no differences between CPF and control mice were found when assessed under basal condition (saline administration), but both male and female CPF mice missed to show the typical behavioural effects of the 5HTT inhibitor fluvoxamine. During maternal aggression, CPF females showed lower propensity to and intensity of aggressive behaviour, together with mild decreased responsiveness to fluvoxamine administration.

Conclusions

Overall, the present results confirm a specific and sex-dependent vulnerability of affective/emotional domains to developmental CPF exposure. Furthermore, data provide clear indication on the disrupting effects of prenatal CPF on serotoninergic transmission.     

Prevalence of efflux-mediated ciprofloxacin and levofloxacin resistance in recent clinical isolates of Pseudomonas aeruginosa and its reversal by the efflux pump inhibitors 1-(1-naphthylmethyl)-piperazine and phenylalanine-arginine-β-naphthylamide

To assess the prevalence of efflux-driven fluoroquinolone (FQ) resistance in recent clinical isolates of Pseudomonas aeruginosa, a worrisome and often hospital-acquired pathogen, 115 unique strains were collected over a 5-month period, of which 27 and 33 had decreased susceptibility to ciprofloxacin (CIP) and levofloxacin (LVX), respectively. The MIC₅₀ (minimum inhibitory concentration for 50% of the organisms) was 16 μg/mL for both FQs. The efflux pump inhibitors (EPIs) phenylalanine-arginine-β-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine (NMP) were then used to evaluate their efficacy in reducing CIP and LVX MICs. NMP did not significantly modify CIP MICs, whilst PAβN resulted in MIC₅₀ values of 2 μg/mL and 0.125 μg/mL for CIP and LVX, respectively. With the addition of PAβN, susceptibility to CIP and LVX was recovered in 6 (22.2%) and 31 (93.9%) strains, respectively. The best combination to reverse FQ resistance in this set of strains was LVX with PAβN. The results of this study show that the effect of an EPI is not only dependent on the species on which it is used but also on the molecule associated with it. Therefore, the design of an EPI equally efficient on all resistance-nodulation-cell division (RND) efflux pumps appears to be difficult and, from a practical point of view, if an EPI is developed for clinical use, the efficiency of its combination with a definite molecule should be assessed carefully against a wide range of clinical isolates to evaluate the real benefit of this combination.     

Heroin increases the density and sensitivity of platelet α2-adrenoceptors in human addicts

The density of platelet ₂-adrenoceptors, quantified by means of the binding of [³H]clonidine, and the aggregation response induced by adrenaline, was investigated in ten heroin addicts. The number of binding sites for [³H]clonidine was significantly increased during heroin abuse. Concomitantly, there was a potentiation of the adrenaline-induced platelet aggregation, which suggested that continuous heroin use (opiate dependence) is associated with supersensitive platelet ₂-adrenoceptors in human addicts. Spontaneous heroin withdrawal further increased in the same addicts the density of platelet ₂-adrenoceptors. These results suggest that platelet ₂-adrenoceptors may be used as a model to study receptor mechanisms of opiate dependence and withdrawal in humans     

1974–2014: Reflections on the evolution of clinical pharmacology in the past 40 years and a message to our readers

Hypersensitivity reactions including anaphylaxis have been reported for nearly all classes of therapeutic reagents and these reactions can occur within minutes to hours of exposure. These reactions are unpredictable, not directly related to dose or the pharmacological action of the drug and have a relatively high mortality risk. This review will focus on the clinical presentation, immune mechanisms, diagnosis and prevention of the most serious form of immediate onset drug hypersensitivity reaction, anaphylaxis. The incidence of drug-induced anaphylaxis deaths appears to be increasing and our understanding of the multiple and complex reasons for the unpredictable nature of anaphylaxis to drugs is also expanding. This review highlights the importance of enhancing our understanding of the biology of the patient (i.e. immune response, genetics) as well as the pharmacology and chemistry of the drug when investigating, diagnosing and treating drug hypersensitivity. Misdiagnosis of drug hypersensitivity leads to substantial patient risk and cost. Although oral provocation is often considered the gold standard of diagnosis, it can pose a potential risk to the patient. There is an urgent need to improve and standardize diagnostic testing and desensitization protocols as other diagnostic tests currently available for assessment of immediate drug allergy are not highly predictive.