In silico molecular docking study of natural compounds on wild and mutated epidermal growth factor receptor

The role played by overexpression of tyrosine kinase epidermal growth factor receptor (EGFR), the transmembrane receptor central to numerous cellular processes comprising cell migration, adhesion, apoptosis, and cell proliferation, has been highlighted in various cancers such as prostate, breast, lung, and ovarian cancers as well as in mutations in the EGFR kinase domain. Although many therapeutic approaches have targetted EGFR, the mutations occurring in the EGFR kinase domain including L858 EGFR and T790/L858R had led to the amplification of EGFR signals, consequently leading to increased cell proliferation and cell growth. The strategies involving the inhibition of EGFR L858 and T790M have been accredited with limited achievement in addition to being associated with unwanted adverse effects as a result of crosstalk of wild-type EGFR. All current EGFR tyrosine kinase inhibitors have been identified as ATP competitive inhibitors of wild-type EGFR possessing aniline and quinazoline moiety on the ligands skeleton. Our results obtained by performing molecular docking study on Maestro 9.3 molecular docking suite indicated that CID5280343 possesses better energy conformation against wild-type EGFR as well as two mutated EGFR. Moreover, it was discovered in this study that the natural compounds CID72276, CID5280445, CID441794, and CID72277 and InterBioScreen’s library STOCK1N-78657, STOCK1N-78976, and STOCK1N-78847 have better binding conformation against gatekeeper T790M mutated EGFR concluded to be brought about by means of flexible ligands/receptor-based molecular docking protocol. Miraculous features of these compounds are their various pharmacokinetic and pharmacodynamic parameters which were found to be satisfactory as drug-like molecules. This molecular docking study also summarizes docking free energy, protein–ligands interaction profile, and pharmacokinetic and pharmacodynamic parameter of lead molecules which were tremendously helpful in enhancing the activity of these natural compounds against EGFR.     

Targeting EGFR mutants with non-cognate kinase inhibitors in non-small cell lung cancer

Considering that the human protein kinase family members share high conservation in both primary sequence and advanced structure, and a large number of small-molecule inhibitors have already been developed to target these different members, we herein are interested in whether certain existing inhibitors that were originally designed for other (cognate) kinases can also bind efficiently to (non-cognate) epidermal growth factor receptor (EGFR) mutants and simultaneously lie low affinity to wild-type EGFR. To explore this notion, a structure-based quantitative structure–activity relationship model was derived from 77 crystal structure-solved, affinity-known kinase–inhibitor complexes. We employed this model to profile a systematic interaction map of 2 cognate and 11 non-cognate kinase inhibitors with wild-type EGFR and its several important NSCLC-related mutants. As might be expected, the cognate inhibitors exhibited generally high affinity to both wild-type and mutant EGFR, while most non-cognate inhibitors have low binding potency for EGFR. However, few combinations of non-cognate inhibitors and EGFR mutants, such as TAE684 and T790M, SKI606 and L858R, and R406 and T790M, show a favorable interaction as compared to the same inhibitors with wild-type EGFR. A further kinase assay was performed to determine the inhibitory activities of compound TAE684, originally developed as a ALK kinase inhibitor, against wild-type and T790M mutant EGFR; it is revealed that the TAE684 inhibited the mutant with ~tenfold higher potency than the wild-type enzyme. This work would help to establish a new strategy for the new use of an old drug.     

MDMA administration during adolescence exacerbates MPTP-induced cognitive impairment and neuroinflammation in the hippocampus and prefrontal cortex

Rationale

We have recently shown that chronic exposure to 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) of adolescent mice exacerbates dopamine neurotoxicity and neuroinflammatory effects elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the substantia nigra and striatum at adulthood.

Objectives

The present study investigated whether the amplification of MPTP effects by previous treatment with MDMA extends to the limbic and cortical regions and consequently affects cognitive performance.

Methods

Mice received MDMA (10 mg/kg, twice a day/twice a week) for 9 weeks, followed by MPTP (20 mg/kg?×?4 administrations), starting 2 weeks after MDMA discontinuation. Complement type 3 receptor (CD11b) and glial fibrillary acidic protein (GFAP) were evaluated by immunohistochemistry in both the hippocampus and the medial prefrontal cortex (mPFC) to measure microglia and astroglia activation. These neurochemical evaluations were paired with an assessment of cognitive performance by means of the novel object recognition (NOR) and spontaneous alternation tasks.

Results

MPTP administration to MDMA-pretreated mice elicited a stronger activation of CD11b and GFAP in both the hippocampus and the mPFC compared with either substance administered alone. Furthermore, NOR performance was lower in MDMA-pretreated mice administered MPTP compared with mice that received either substance alone.

Conclusions

These results demonstrate that MDMA–MPTP negative interactions extend to the limbic and cortical regions and may result in cognitive impairment, providing further evidence that exposure to MDMA may amplify the effects of later neurotoxic insults.     

Developmental alterations in anxiety and cognitive behavior in serotonin transporter mutant mice

Rationale

A promoter variant of the serotonin transporter (SERT) gene is known to affect emotional and cognitive regulation. In particular, the “short” allelic variant is implicated in the etiology of multiple neuropsychiatric disorders. Heterozygous (SERT^+/?) and homozygous (SERT^?/?) SERT mutant mice are valuable tools for understanding the mechanisms of altered SERT levels. Although these genetic effects are well investigated in adulthood, the developmental trajectory of altered SERT levels for behavior has not been investigated.

Objectives

We assessed anxiety-like and cognitive behaviors in SERT mutant mice in early adolescence and adulthood to examine the developmental consequences of reduced SERT levels. Spine density of pyramidal neurons was also measured in corticolimbic brain regions.

Results

Adult SERT^?/? mice exhibited increased anxiety-like behavior, but these differences were not observed in early adolescent SERT^?/? mice. Conversely, SERT^+/? and SERT^?/? mice did display higher spontaneous alternation during early adolescence and adulthood. SERT^+/? and SERT^?/? also exhibited greater neuronal spine densities in the orbitofrontal but not the medial prefrontal cortices. Adult SERT^?/? mice also showed an increased spine density in the basolateral amygdala.

Conclusions

Developmental alterations of the serotonergic system caused by genetic inactivation of SERT can have different influences on anxiety-like and cognitive behaviors through early adolescence into adulthood, which may be associated with changes of spine density in the prefrontal cortex and amygdala. The altered maturation of serotonergic systems may lead to specific age-related vulnerabilities to psychopathologies that develop during adolescence.     

PACE: Pharmacists use the power of communication in paediatric asthma

Background Paediatric asthma is a public health burden in Australia despite the availability of national asthma guidelines. Community pharmacy interventions focusing on paediatric asthma are scarce. Practitioner Asthma Communication and Education (PACE) is an evidence-based program, developed in the USA for general practice physicians, aimed at addressing the issues of poor clinician-patient communication in the management of paediatric asthma. This program has been shown to improve paediatric asthma management practices of general practitioners in the USA and Australia. The development of a PACE program for community pharmacists will fill a void in the current armamentarium for pharmacist-patient care. Objectives To adapt the educational program, PACE, to the community pharmacy setting. To test the feasibility of the new program for pharmacy and to explore its potential impact on pharmacists’ communication skills and asthma related practices. Setting Community pharmacies located within the Sydney metropolitan. Method The PACE framework was reviewed by the research team and amended in order to ensure its relevance within the pharmacy context, thereby developing PACE for Pharmacy. Forty-four pharmacists were recruited and trained in small groups in the PACE for Pharmacy workshops. Pharmacists’ satisfaction and acceptability of the workshops, confidence in using communication strategies pre- and post-workshop and self-reported behaviour change post workshop were evaluated. Main Outcome Measure Pharmacist self-reported changes in communication and teaching behaviours during a paediatric asthma consultation. Results All 44 pharmacists attended both workshops, completed pre- and post-workshop questionnaires and provided feedback on the workshops (100 % retention). The participants reported a high level of satisfaction and valued the interactive nature of the workshops. Following the PACE for Pharmacy program, pharmacists reported significantly higher levels in using the communication strategies, confidence in their application and their helpfulness. Pharmacists checked for written asthma self-management plan possession and inhaler device technique more regularly, and provided verbal instructions more frequently to paediatric asthma patients/carers at the initiation of a new medication. Conclusion This study provides preliminary evidence that the PACE program can be translated into community pharmacy. PACE for Pharmacy positively affected self-reported communication and education behaviours of pharmacists. The high response rate shows that pharmacists are eager to expand on their clinical role in primary healthcare.     

Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function

Rationale

Neurosteroids and likely other lipid modulators access transmembrane sites on the GABA_A receptor (GABA_AR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABA_ARs.

Objectives

In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine.

Methods

We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABA_ARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion.

Results

We found no evidence that PIP2 depletion affected modulation of GABA_ARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity.

Conclusions

We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABA_ARs by neurosteroids or related compounds.     

The effect of Eudragit type on BSA-loaded PLGA nanoparticles

Poly(d,l-lactide-co-glycolide) nanoparticle (PLGA NP) have been broadly studied as a carrier for drug delivery system of peptides and proteins. However, negative surface charge of PLGA NP using only PLGA decreases bioavailability under oral administration. In this study, novel carriers for oral delivery system through an additional bioadhesive polymer, Eudragit was introduced. Our purpose is to prepare PLGA NP using bovine serum albumin (BSA) as a model drug and Eudragit and evaluate their physiochemical characteristics, eventually expand to peptide and protein drug such as insulin or exenatide. In this study, PLGA NP were spherical and the size was around 400–500 nm. The encapsulation efficiency (EE) of PLGA NP when prepared with only PLGA was the highest, approximately 95.3 %. The polydispersity index values were low approximately 0.1, which meant their size was regular. In mucoadhesion test, we knew PLGA NP prepared by using Eudragit RS or Eudragit RL had a high affinity to mucin particles through zeta-potential change of mucin particle to cover their surface. Also, PLGA NP did not show cytotoxicity against Caco-2 cells. Especially, BSA-loaded PLGA NP using Eudragit RS 100 prepared had high EE, low polydispersity index, spherical shape having a smooth surface, sustained release profile, non-cytotoxicity and bioadhesive effect.     

Dual and/or selective DNA-PK,PI3K inhibition and isoform selectivity of some new and known 2-amino-substituted-1,3-benzoxazines and substituted-1,3-naphthoxazines

The 2-morpholino-substituted-benzoxazines 7a and 7b were used in the synthesis of 2-morpholino-di-O-benzyl, O-pyridin-2yl, 3-yl and 4yl-methoxy)-1,3-benzoxazines 8a–8d, and N-(2-morpholino-4-oxo-4H-benz[e][1,3]oxazin-7-yl)-N-(pyridin-2-and-3-ylmethyl)acetamides 8e and 8f. The DNA-dependent protein kinase (DNA-PK) and phosphatidylinositol 3-kinase (PI3K) α, β, γ, and δ isoforms were studied for the new compounds 8a–8f and PI3K for the 18 previously synthesized compounds 9–26. The most active DNA-PK inhibitors were the 2-morpholino-O-substituted linear or angular naphthoxazine compounds 18–20 and 21–22 which showed potent and selective DNA-PK activity (IC₅₀ from 0.01 to 2.43 µM) over PI3K. 8-(2-(4-Methylpiperazin-1-yl)ethoxy)-2-morpholino compound 13, and 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy) compound 25 showed selective DNA-PK inhibition. 2-morpholino-8-substituted-benzoxazine 9 (8-ph) and 10–12 (8-(pyridine-2-, 3-, or 4-ylmethoxy) showed high-to-moderate inhibition of PI3K and DNA-PK. A similar pattern for DNA-PK nonselectivity over PI3K was observed for compounds with 7,8-O-bis-substituted 8a, 8c, and 8d. No DNA-PK selectivity over PI3K was observed regardless whether the substitution was phenyl, pyridin-2-ylmethoxy, pyridin-3-ylmethoxy, and pyridin-4-ylmethoxy.     

Phosphorylation of GABAA receptors influences receptor trafficking and neurosteroid actions

Rationale

Gamma-aminobutyric acid type A receptors (GABA_ARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABA_ARs can be localized at post-synaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABA_ARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABA_ARs are typically activated continuously by ambient GABA concentrations and thus mediate tonic inhibition. The tonic inhibitory currents mediated by extrasynaptic GABA_ARs control neuronal excitability and the strength of synaptic transmission. However, the mechanisms by which neurons control the functional properties of extrasynaptic GABA_ARs had not yet been explored.

Objectives

We review GABA_ARs, how they are assembled and trafficked, and the role phosphorylation has on receptor insertion and membrane stabilization. Finally, we review the modulation of GABA_ARs by neurosteroids and how GABA_AR phosphorylation can influence the actions of neurosteroids.

Conclusions

Trafficking and stability of functional channels to the membrane surface are critical for inhibitory efficacy. Phosphorylation of residues within GABA_AR subunits plays an essential role in the assembly, trafficking, and cell surface stability of GABA_ARs. Neurosteroids are produced in the brain and are highly efficacious allosteric modulators of GABA_AR-mediated current. This allosteric modulation by neurosteroids is influenced by the phosphorylated state of the GABA_AR which is subunit dependent, adding temporal and regional variability to the neurosteroid response. Possible links between neurosteroid actions, phosphorylation, and GABA_AR trafficking remain to be explored, but potential novel therapeutic targets may exist for numerous neurological and psychological disorders which are linked to fluctuations in neurosteroid levels and GABA_A subunit expression.     

Zinc Protoporphyrin Polymeric Nanoparticles: Potent Heme Oxygenase Inhibitor for Cancer Therapy

Purpose

Oxidation therapy is an antitumor strategy in which, apoptosis or necrosis is caused by either excess delivery of reactive oxygen species (ROS) as an oxidant or anti-oxidant inhibition. Heme oxygenase (HO) is an anti-oxidant enzyme that plays an important role in cell growth and proliferation. The purpose of this study was to prepare poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with zinc protoporphyrin (ZnPP) to deliver the HO inhibitor into tumor.

Methods

PLGA NPs were prepared using nanoprecipitation technique and their characteristics were optimized by Box-Behnken experimental design. Scanning electron microscopy and in vitro studies consisting of drug release, HO inhibitory effect, cytotoxicity and cellular uptake followed by in vivo biodistribution and blood cytotoxicity were carried out. Internalization of coumerin-6 loaded NPs by PC3 cells was visualized by confocal laser scanning microscopy beside quantitatively analysis.

Results

NPs average size, entrapment efficiency and drug loading were 100.12?±?5.345 nm, 55.6%?±?2.49 and 7.98%?±?0.341 respectively. Equal HO inhibitory effect of NPs compared to free ZnPP was observed. The IC₅₀ value of ZnPP-NPs for PC3 human prostate cancer cells was found to be 2.14?±?0.083 μM.

Conclusion

In conclusion, ZnPP loaded PLGA NPs could exhibit enough HO inhibitory effect against cancer cells to be considered as a promising candidate for cancer treatment investigation.     

Oxidative stress and BDNF as possible markers for the severity of crack cocaine use in early withdrawal

Rationale

An important goal of addiction research is to discover neurobiological markers that could predict the severity of addiction and help to determine appropriate treatment. Brain-derived neurotrophic factor (BDNF) and thiobarbituric acid reactive substances (TBARS) are being related to cerebral plasticity and impairment caused by substance abuse.

Objectives

This study aims to evaluate alteration of TBARS and BDNF levels among crack cocaine users during early drug withdrawal and its relationship to severity of drug use.

Methods

Forty-nine adults crack cocaine users were recruited at a public psychiatric hospital with a specialized addiction treatment unit. Blood sample was collected at intake and discharge for the analysis of TBARS and BDNF measures. Information about drug use was assessed by the Addiction Severity Index 6th Version (ASI-6). Detailed information about crack cocaine use was obtained through the “Profile of the crack cocaine user.” Severity of crack use was estimated using information from age of first crack use, years of crack use, and crack rocks used in the previous 30 days.

Results

There is a positive correlation between TBARS levels and severity of crack cocaine use (R?=?0.304, p?=?0.04) and a negative correlation between BDNF and severity of crack cocaine use (R?=??0.359, p?=?0.01) at discharge. Also, we found an inverse correlation between TBARS and BDNF levels (R?=??0.294, p?=?0.004) at discharge.

Conclusions

Our findings suggest that BDNF and TBARS could be possible markers for the severity of drug use. Further studies may show how those markers could be related to staging, prognosis, and treatment in crack cocaine dependence.     

Ultrasound Enhanced PEI-Mediated Gene Delivery Through Increasing the Intracellular Calcium Level and PKC-δ Protein Expression

Purpose

Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. It has also been shown that combining ultrasound (US) with PEI could enhance and prolong in vitro and in vivo transgene expression. However, the role US in the enhancement of PEI uptake is poorly understood. This study investigates the impact of US on PEI-mediated gene transfection.

Methods

Specific endocytosis pathway siRNA, including clathrin HC siRNA, caveolin-1 siRNA and protein kinase C-delta (PKC-δ) siRNA, are used to block the corresponding endocytosis pathways prior to the transfection of luciferase DNA/PEI polyplexes to cultured cells by 1-MHz pulsed US with ultrasound contrast agent SonoVue®.

Results

Transgene expression was found not to be enhanced by US treatment in the presence of the PKC-δ siRNA. We further demonstrated that PKC-δ protein could be enhanced at 6 h after US exposure. Moreover, intracellular calcium levels were found to be significantly increased at 3 h after US exposure, while transgene expressions were significantly reduced in the presence of calcium channel blockers both in vitro and in vivo.

Conclusions

Our results suggest that US enhanced PEI-mediated gene transfection specifically by increasing PKC-δ related fluid phase endocytosis, which was induced by increasing the intracellular calcium levels.     

MDMA alters emotional processing and facilitates positive social interaction

Background

±3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) produces “prosocial” effects, such as feelings of empathy and closeness, thought to be important to its abuse and its value in psychotherapy. However, it is not fully understood how MDMA alters basic emotional processes to produce these effects, or whether it produces corresponding changes in actual social behavior. Here, we examined how MDMA affects perceptions of and responses to emotional expressions, and tested its effects on behavior during a social interaction. We also examined whether MDMA’s prosocial effects related to a measure of abuse liability.

Methods

Over three sessions, 36 healthy volunteers with previous ecstasy use received MDMA (0.75, 1.5 mg/kg) and placebo under double-blind conditions. We measured (i) mood and cardiovascular effects, (ii) perception of and psychophysiological responses to emotional expressions, (iii) use of positive and negative words in a social interaction, and (iv) perceptions of an interaction partner. We then tested whether these effects predicted desire to take the drug again.

Results

MDMA slowed perception of angry expressions, increased psychophysiological responses to happy expressions, and increased positive word use and perceptions of partner empathy and regard in a social interaction. These effects were not strongly related to desire to take the drug again.

Conclusions

MDMA alters basic emotional processes by slowing identification of negative emotions and increasing responses to positive emotions in others. Further, it positively affects behavior and perceptions during actual social interaction. These effects may contribute to the efficacy of MDMA in psychotherapy, but appear less closely related to its abuse potential.     

The role of ovarian hormone-derived neurosteroids on the regulation of GABAA receptors in affective disorders

Rationale

Neuroactive derivatives of steroid hormones, neurosteroids, can act on GABA_A receptors (GABA_ARs) to potentiate the effects of GABA on these receptors. Neurosteroids become elevated to physiologically relevant levels under conditions characterized by increased steroid hormones. There is considerable evidence for plasticity of GABA_ARs associated with altered levels of neurosteroids which may counteract the fluctuations in the levels of these allosteric modulators.

Objectives

The objective of this review is to summarize the current literature on GABA_AR plasticity under conditions characterized by alterations in neurosteroid levels, such as over the estrous cycle, during puberty, and throughout pregnancy and the postpartum period.

Results

The expression of specific GABA_AR subunits is altered over the estrous cycle, at puberty, and throughout pregnancy and the postpartum period. Inability to regulate δ subunit-containing GABA_ARs throughout pregnancy and the postpartum period is associated with depression-like behavior restricted to the postpartum period.

Conclusions

GABA_AR plasticity associated with alterations in neurosteroid levels represents a homeostatic compensatory mechanism to maintain an ideal level of inhibition to offset the potentiating effects of neurosteroids on GABAergic inhibition. Failure to properly regulate GABA_ARs under conditions of altered neurosteroid levels may increase vulnerability to mood disorders, such as premenstrual syndrome (PMS), premenstrual dysphoric disorder (PMDD), and postpartum depression.     

ABC Transporters in Multi-Drug Resistance and ADME-Tox of Small Molecule Tyrosine Kinase Inhibitors

The past decade has seen tremendous efforts in the research and development of new chemotherapeutic drugs using target-based approaches. These efforts have led to the discovery of small molecule tyrosine kinase inhibitors (TKIs). Following the initial approval of imatinib by the US FDA in 2001, more than 15 TKIs targeting different tyrosine kinases have been approved, and numerous others are in various phases of clinical evaluation. Unlike conventional chemotherapy that can cause non-discriminating damage to both normal and cancerous cells, TKIs attack cancer-specific targets and therefore have a more favorable safety profile. However, although TKIs have had outstanding success in cancer therapy, there has been increasing evidence of resistance to TKIs. The enhanced efflux of TKIs by ATP-binding cassette (ABC) transporters over-expressed in cancer cells has been found to be one such important resistance mechanism. Another major drawback of TKI therapies that has been increasingly recognized is the extensive inter-individual pharmacokinetic variability, in which ABC transporters seem to play a major role as well. This review covers recent findings on the interactions of small molecule TKIs with ABC transporters. The effects of ABC transporters on anticancer efficacy and the absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) of the small molecule TKIs are summarized in detail. Since TKIs have been found to not only serve as substrates of ABC transporters, but also as modulators of these proteins via inhibition or induction, their influence upon ABC transporters and potential role on TKI-drug interactions are discussed as well.     

3,4-Methylenedioxymethamphetamine (MDMA – Ecstasy) Decreases Neutrophil Activity Through the Glucocorticoid Pathway and Impairs Host Resistance to Listeria Monocytogenes Infection in Mice

Ecstasy is the popular name of the abuse drug 3,4-methylenedioxymethamphetamine (MDMA) that decreases immunity in animals. The mechanisms that generate such alterations are still controversial. Seven independent pharmacological approaches were performed in mice to identify the possible mechanisms underlying the decrease of neutrophil activity induced by MDMA and the possible effects of MDMA on host resistance to Listeria monocytogenes. Our data showed that MDMA (10 mg kg^?1) administration decreases NFκB expression in circulating neutrophils. Metyrapone or RU-486 administration prior to MDMA treatment abrogated MDMA effects on neutrophil activity and NFκB expression, while 6-OHDA or ICI-118,551 administration did not. As MDMA treatment increased the plasmatic levels of adrenaline and noradrenaline, propranolol pre-treatment effects were also evaluated. Propranolol suppressed both MDMA-induced increase in corticosterone serum levels and its effects on neutrophil activity. In a L. monocytogenes experimental infection context, we showed that MDMA: induced myelosuppression by decreasing granulocyte-macrophage hematopoietic progenitors (CFU-GM) in the bone marrow but increased CFU-GM in the spleen; decreased circulating leukocytes and bone marrow cellularity and increased spleen cellularity; decreased pro-inflammatory cytokine (IL-12p70, TNF, IFN-γ, IL-6) and chemokine (MCP-1) production 24 h after the infection; increased the production of pro-inflammatory cytokines and chemokines 72 h after infection and decreased IL-10 levels at all time points analyzed. It was proposed that MDMA immunosuppressive effects on neutrophil activity and host resistance to L monocytogenes rely on NFκB signaling, being mediated by HPA axis activity and corticosterone.     

Inhibition of human drug-metabolising cytochrome P450 and UDP-glucuronosyltransferase enzyme activities in vitro by uremic toxins

Objective

To investigate the potential inhibitory effects of uremic toxins on the major human hepatic drug-metabolising cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes in vitro.

Methods

Benzyl alcohol, p-cresol, indoxyl sulfate, hippuric acid and a combination of the four uremic toxins were co-incubated with human liver microsomes and selective probe substrates for the major human drug-metabolising CYP and UGT enzymes. The percentage of enzyme inhibition was calculated by measuring the rates of probe metabolite formation in the absence and presence of the uremic toxins. Kinetics studies were conducted to evaluate the K _i values and mechanism(s) of the inhibition of CYP2E1, CYP3A4, UGT1A1 and UGT1A9 by p-cresol.

Results

The individual uremic toxins inhibited CYP and UGT enzymes to a variable extent. p-Cresol was the most potent individual inhibitor, producing >50 % inhibition of CYP2E1, CYP3A4, UGT1A1, UGT1A9 and UGT2B7 at a concentration of 100 μM. The greatest inhibition was observed with UGT1A9. p-Cresol was shown to be an uncompetitive inhibitor of UGT1A9, with unbound K _i values of 9.1 and 2.5 μM in the absence and presence of bovine serum albumin (BSA), respectively. K _i values for p-cresol inhibition of human liver microsomal CYP2E1, CYP3A4 and UGT1A1 ranged from 43 to 89 μM. A combination of the four uremic toxins produced >50 % decreases in the activities of CYP1A2, CYP2C9, CYP2E1, CYP3A4, UGT1A1, UGT1A9 and UGT2B7.

Conclusions

Uremic toxins may contribute to decreases in drug hepatic clearance in individuals with kidney disease by inhibition of hepatic drug-metabolising enzymes.     

Insight into Mechanisms of Cellular Uptake of Lipid Nanoparticles and Intracellular Release of Small RNAs

Purpose

Understanding mechanisms of cellular uptake and intracellular release would enable better design of nanocarriers for delivery of nucleic acids such as siRNA and microRNA (miRNA).

Method

In this study, we investigated cellular pharmacokinetics of siRNA by co-encapsulating fluorescently labeled siRNA and molecular beacon (MB) in four different formulations of cationic lipid nanoparticles (LNPs). A miRNA mimic was also used as a probe for investigating cellular pharmacokinetics, which correlated well with RNAi activities.

Results

We tried to find the best LNP formulation based on the combination of DOTMA and DODMA. When the DOTMA/DODMA ratio was at 5/40, the LNP containing a luciferase siRNA produced the highest gene silencing activity. The superior potency of DOTMA/DODMA could be attributed to higher uptake and improved ability to facilitate siRNA release from endosomes subsequent to uptake.

Conclusions

Our findings may provide new insights into RNAi transfection pathways and have implications on cationic LNP design.     

Beta-2 adrenergic receptors mediate stress-evoked reinstatement of cocaine-induced conditioned place preference and increases in CRF mRNA in the bed nucleus of the stria terminalis in mice

Rationale

Understanding the mechanisms responsible for stress-induced relapse is important for guiding treatment strategies aimed at minimizing the contribution of stress to addiction. Evidence suggests that these mechanisms involve interactions between noradrenergic systems and the neuropeptide corticotropin-releasing factor (CRF).

Objectives

The interaction between β-adrenergic receptors (ARs) and CRF as it relates to the reinstatement of cocaine-conditioned reward in response to a stressor was examined in mice. We hypothesized that β₂-ARs are required for stress-induced activation of CRF pathways responsible for reinstatement.

Methods

Stress-induced relapse was examined based on the re-establishment of cocaine-induced conditioned place preference (CPP; 4?×?15 mg/kg cocaine, i.p.) after extinction using forced swim (6 min at 22 °C) or an injection of the β₂-AR agonist, clenbuterol (4 mg/kg, i.p.). The CRF-R1 antagonist antalarmin (10 mg/kg, i.p.) or the β₂-AR antagonist ICI-118,551 (1 mg/kg, i.p.) were given 30 min prior to reinstating stimuli. Quantitative PCR was conducted in dissected bed nucleus of the stria terminalis (BNST) and amygdala, putative sources of CRF that contribute to reinstatement, to examine the effects of ICI-118,551 on swim-induced increases in CRF messenger RNA (mRNA) in mice with a cocaine history.

Results

Pretreatment with ICI-118,551 or antalarmin blocked swim-induced reinstatement of CPP. Reinstatement by clenbuterol was also blocked by antalarmin. ICI-118,551 pretreatment prevented swim-induced increases in CRF mRNA in the BNST. Effects in the amygdala were not observed.

Conclusions

These findings indicate that, during stress, norepinephrine, via β₂-ARs, either directly or indirectly activates CRF-releasing neurons in the BNST that interface with motivational neurocircuitry to induce reinstatement of cocaine-conditioned reward.